WO2002055547A9 - Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes - Google Patents

Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes

Info

Publication number
WO2002055547A9
WO2002055547A9 PCT/US2002/000869 US0200869W WO02055547A9 WO 2002055547 A9 WO2002055547 A9 WO 2002055547A9 US 0200869 W US0200869 W US 0200869W WO 02055547 A9 WO02055547 A9 WO 02055547A9
Authority
WO
WIPO (PCT)
Prior art keywords
sfrp
peptide
seq
ala
group
Prior art date
Application number
PCT/US2002/000869
Other languages
English (en)
Other versions
WO2002055547A3 (fr
WO2002055547A2 (fr
Inventor
Jeffrey S Rubin
Aykut Uren
Nicole Joy Horwood
Matthew Todd Gillespie
Brian K Kay
Bernard Weisblum
Original Assignee
Us Gov Health & Human Serv
St Vincents Inst Med Res
Jeffrey S Rubin
Aykut Uren
Nicole Joy Horwood
Matthew Todd Gillespie
Brian K Kay
Bernard Weisblum
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP02707454A priority Critical patent/EP1387854B1/fr
Priority to AU2002241859A priority patent/AU2002241859B2/en
Priority to ES02707454T priority patent/ES2385666T3/es
Priority to US10/466,136 priority patent/US7488710B2/en
Priority to DK02707454.1T priority patent/DK1387854T3/da
Priority to CA2434672A priority patent/CA2434672C/fr
Application filed by Us Gov Health & Human Serv, St Vincents Inst Med Res, Jeffrey S Rubin, Aykut Uren, Nicole Joy Horwood, Matthew Todd Gillespie, Brian K Kay, Bernard Weisblum filed Critical Us Gov Health & Human Serv
Priority to JP2002556615A priority patent/JP4029041B2/ja
Priority to AT02707454T priority patent/ATE550345T1/de
Publication of WO2002055547A2 publication Critical patent/WO2002055547A2/fr
Publication of WO2002055547A9 publication Critical patent/WO2002055547A9/fr
Publication of WO2002055547A3 publication Critical patent/WO2002055547A3/fr
Priority to US12/019,567 priority patent/US8334260B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0643—Osteoclasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • A61P5/22—Drugs for disorders of the endocrine system of the parathyroid hormones for decreasing, blocking or antagonising the activity of calcitonin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This disclosure relates to osteoclast differentiation, specifically to a peptide motif and proteins containing the motif that are capable of binding to secreted Frizzled-related protein family members.
  • Bone remodeling a process responsible for the continuous renewal of the adult human skeleton, is carried out by osteoclasts and osteoblasts, two specialized cell types that originate from hematopoietic and mesenchymal progenitors of the bone marrow, respectively.
  • a continuous and orderly supply of these cells is essential for skeletal homeostasis, as increased or decreased production of osteoclasts or osteoblasts and/or changes in the rate of their apoptosis are largely responsible for the imbalance between bone resorption and formation that underlies several systemic or localized bone diseases.
  • Enhanced osteoclast activity plays a major role in the pathogenesis of postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, hyperparathyroidism, rheumatoid arthritis, periodontitis, and hypercalcemia of malignancy.
  • These clinical problems are associated with significant morbidity or mortality, and affect more than 10 million patients in the United States.
  • only a limited number of agents that inhibit osteoclast formation or bone resorption are available and for most their mechanisms of action are unknown.
  • many of these agents have significant side effects that limit their utility.
  • osteoclast activity plays a major role in the pathogenesis of osteopetrosis, Albright's osteodystrophy, and achondroplasia, for which there is no specific therapy.
  • bone disorders Identification of the mechanisms involved in bone disorders is crucial for the understanding of bone physiology. While numerous genes and gene families (and the polypeptides encoded by them) that participate in the regulation of bone cells have been identified and cloned, their functions have not been clearly delineated due to the complexities of the bone formation pathways. A great need exists for the definitive identification of targets for the treatment of bone disorders, including bone resorption disorders such as postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, rheumatoid arthritis, hypercalcemia of malignancy, osteopetrosis, Albright's osteodystrophy, and achondroplasia.
  • bone resorption disorders such as postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, rheumatoid arthritis, hypercalcemia of malignancy, osteopetrosis, Albright's osteodystrophy, and
  • the sFRP-1 binding peptide is a purified peptide.
  • the peptide is selected from the group consisting of: (a) the amino acid sequence shown in SEQ ID NO: 9; (b) at least one conservative amino acid substitution of the amino acid sequence shown in (a); and (c) an amino acid sequence that shares at least 80% sequence identity with the sequence shown in (a), wherein the protein retains the ability to bind to sFRP.
  • the peptide has a sequence as shown in the formula:
  • [Rl]x - R2 - R3 - R4- R5 - R6 - R7 - R8 - [R9]y wherein x and y are integers independently selected from the group 0 or 1; R3 is selected from the group Val (V), Ala (A) or conservative substitutions therefor; R4 is selected from the group consisting of Asp (D), Ala (A) or conservative substitutions therefor; R5 is selected from the group consisting of Gly (G), Ala (A) or conservative substitutions therefor; R6 is selected from the group consisting of Arg (R) Ala (A) or conservative substitutions therefor; R7 is selected from the group consisting of Trp (W), Ala (A) or conservative substitutions therefor. Nucleic acids encoding these peptides are provided, as are vectors containing the nucleic acids and host cells transformed with these vectors. Methods for screening for agents that interfere with or mimic the interaction of these peptides and sFRP are also disclosed.
  • a method for enhancing osteoclast differentiation.
  • the method includes admMstering a therapeutically effective amount of the purified peptides disclosed herein (or effective fragments, fusions or mimetics) to a subject in order to enhance osteoclast differentiation.
  • a method for inhibiting osteoclast formation in a subject includes administering to the subject a therapeutically effective amount of sFRP-1 (SEQ ID NO: 3), fragments of SEQ ID NO: 3, or fusions or variants of SEQ ID NO: 3, to a subject, wherein the polypeptide binds to a RANKL molecule as set forth as GenBank Accession No. AF013171, GenBank Accession No. AF019047, or GenBank Accession No. AF053712, or another TNF family member.
  • a method for modulating T cell activity.
  • the method includes administering a therapeutically effective amount of the purified sFRP-1 -binding peptides disclosed herein to a subject in order to modulate T cell activity.
  • FIG. 1 is a graph showing the results from A-C2 (SEQ ID NO: 14)/AP (alkaline phosphatase) fusion protein binding to sFRP-1 (SEQ ID NO: 3). Broths from separate bacterial colonies infected with phage expressing the A-C2/AP chimera were incubated in ELISA wells coated with sFRP-1 (and subsequently blocked with BSA) or BSA alone.
  • A-C2 SEQ ID NO: 14
  • AP alkaline phosphatase
  • Each of the broths (identified as I- 1, 1-2, 1-6, 1-12, 1-13 and 1-14) contained AP activity, as measured by reaction with pNPP and color development at 405 nm, that bound specifically to the sFRP-1 (SEQ ID NO: 3) -coated wells (gray bars) as compared to the wells coated only with BSA (black bars). Each sample was tested singly; this is representative of several experiments.
  • FIG. 2 is a graph showing the results from a competitive binding assay of A-C2/AP (SEQ ID NO: 14) and the 12-mer peptides A-C2 (SEQ ID NO: 14) and A-F7 (SEQ ID NO: 12) to either BSA or sFRP-1 (SEQ ID NO: 3).
  • FIG. 3 is a graph showing the results from binding assays of alanine substituted A-C2 (SEQ ID NO: 14) and A-F7(SEQ ID NO: 12), were preincubated for 30 minutes at the indicated concentrations with bacterial broth containing A-C2/AP chimera prior to addition to ELISA wells coated with sFRP-1 (A-C2 open squares and A-F7 open diamonds) or BSA (A-C2 open circles and A-F7 open triangles). Samples were tested in triplicate, and results are shown as the mean +/-S.D. This is representative of three experiments.
  • FIG. 3 is a graph showing the results from binding assays of alanine substituted A-C2 (SEQ ID NO: 14) and A-F7(SEQ ID NO: 12), were preincubated for 30 minutes at the indicated concentrations with bacterial broth containing A-C2/AP chimera prior to addition to ELISA wells coated with sFRP-1 (A-C2 open square
  • FIG. 4 is a set of diagrams showing a calorimetric analysis of the interaction between AC2 peptide and sFRP-1.
  • FIG 4 A is a tracing and plot showing the heat generated when aliquots of AC2 solution were added to a chamber containing sFRP-1 dissolved in PBS.
  • FIG. 4B is a tracing and plot of the heat generated in the corresponding PBS control. Based on the amount of heat released, various parameters of the binding reaction were calculated, including the enthalpy ( ⁇ H) and dissociation constant, Kd.
  • FIG. 5 is a graph showing the results from ELISA binding assays using soluble RANKL (sRANKL) and sFRP-1 (SEQ ID NO: 3).
  • the open diamond represents sRANKL binding to sFRP-1, and the plus symbol "+” represents sRANKL binding to bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • FIG. 6 is a set of graphs showing that sFRP-1 inhibits osteoclast formation in two different experimental models.
  • FIG. 6A is a graph showing results from co-culture experiments in which primary osteoblasts and bone marrow were incubated with varying concentrations of sFRP-1. Subsequently, wells were stained to determine the number of TRAP+ multinucleated cells (MNC). Results show that as the concentration of sFRP-1 increases, osteoclast maturation decreases (as is evident by decrease in TRAP+). The results shown are the mean +/- S.D. of quadruplicate measurements.
  • FIG. 6A is a graph showing results from co-culture experiments in which primary osteoblasts and bone marrow were incubated with varying concentrations of sFRP-1. Subsequently, wells were stained to determine the number of TRAP+ multinucleated cells (MNC). Results show that as the concentration of sFRP-1 increases, osteoclast maturation decreases (as is
  • 6B is a graph showing the results from experiments in which adult spleen cells were treated with RANKL, macrophage colony stimulating factor (M-CSF), and various concentrations of sFRP-1 (SEQ ID NO: 3). Subsequently, wells were stained to determine the number of TRAP+ multinucleated cells (MNC). The data presented is the mean +/- S.D. of quadruplicate measurements. Results show that as the concentration of sFRP-1 increases osteoclast maturation decreases.
  • M-CSF macrophage colony stimulating factor
  • FIG. 7 is a pair of graphs showing that sFRP-1 antiserum stimulates osteoclast formation in co-cultures of primary osteoblasts and adult spleen cells.
  • FIG. 7A is a graph showing that sFRP-1 specific antibody binding to sFRP-1 causes an increase in osteoclast formation.
  • Osteoclast formation as measured by TRAP+ staining, was assessed in co-cultures of primary osteoblasts and adult spleen cells without hormonal supplements or with suboptimal doses of l ⁇ ,25(OH 2 ) vitamin D 3 (10 "10 M) and dexamethasone (10 "9 M) in the presence or absence of purified immunoglobulin ( ⁇ 2 ⁇ g/mL) from a rabbit immunized with recombinant sFRP-1.
  • the results are the mean +/- S.D. of mononucleated and multinucleated TRAP+ cells detected in quadruplicate samples after 7 days in culture.
  • 7B is a graph showing that sFRP-1 specific antibodies bind to sFRP-1 and cause an increase in osteoclast formation in the presence of optimal doses of l ⁇ ,25(OH 2 ) vitamin D 3 (10 "s M) and prostaglandin E2 (PGE2) 10 “7 M.
  • Osteoclast formation as measured by TRAP+ staining, was assessed in co-cultures of primary osteoblasts and adult spleen cells without hormonal supplements or with optimal doses of l ⁇ ,25(OH 2 ) vitamin D 3 (10 "8 M) and prostaglandin E2 (PGE2, 10 "7 M) in the presence or absence of purified immunoglobulin ( ⁇ 1 ⁇ g/mL) from a rabbit immunized with recombinant sFRP-1.
  • the results are the mean +/- S.D. of mononucleated and multinucleated TRAP+ cells detected in quadruplicate samples after 7 days in culture.
  • FIG. 8 is a graph showing that A-C2 peptide stimulates osteoclast formation in co-cultures of osteoblasts and adult spleen cells. Osteoclast formation in response to optimal doses of l ⁇ ,25(OH 2 ) vitamin D 3 (10 "8 M) and PGE2 (10 "7 M) was not further enhanced by the concomitant addition of the netrin homology domain (NHD) domain of sFRP-1 (SEQ ID NO: 13; 5 ⁇ g/mL), but it was markedly stimulated by simultaneous incubation with the A-C2 peptide (SEQ ID NO: 14; 5 ⁇ g/mL).
  • NBD netrin homology domain
  • FIG. 9 is a graph showing the results from an experiment in which A-C2 (SEQ ID NO: 14) was incubated for various time periods with adult spleen cells.
  • Group 1 was the control that did not contain A-C2 (SEQ ID NO: 14) peptide.
  • Group 2 was treated with A-C2 (SEQ ID NO: 14) from day 0-3, group 4 was treated with A-C2 (SEQ ID NO: 14) from day 4-7, group 4 was treated with A-C2 (SEQ ID NO: 14) from day 7-10, and group 5 was treated with A-C2 (SEQ ID NO: 14) from day 0- 10. All treatment groups received RANKL at 50 ng/mL and M-CSF at 25 ng/mL.
  • FIGS. 10A and 10B are graphs depicting the results from A-C2 (SEQ ID NO: 14) incubation with adult spleen cells containing T cells (FIG. 10A) and spleen cells without T cells (FIG. 14B): T cells were immunomagnetically separated from the spleen cells. Osteoclast formation was induced by RANKL (50 ng/mL) and M-CSF (25 ng/mL) and assessed by counting TRAP+ multinucleated cells after nine days of culture. Osteoclast formation was measured in adult spleen cell cultures (FIG.
  • FIGS. 11A and 11B are graphs depicting the A-C2 (SEQ ID NO: 14) stimulation of
  • TRAP+ multinucleated cell differentiation in RAW264.7 (TIB-71) cell cultures.
  • Group 1 was the positive control that contained 50 ng/mL RANKL.
  • Groups 2, 3, and 4 contained 50 ng/mL RANKL and either 5 ⁇ g/mL, 1 ⁇ g/mL, and 0.5 ⁇ g/mL of A-C2 (SEQ JD NO: 14) respectively. Stimulation was observed when T cells were added to the cultures (FIG. 15 A) as compared to when T cells were not added to the cultures (FIG. 15B).
  • FIG. 12 is a graph of the binding avidity of several sFRP-1 deletion mutants for RANKL in ELISA experiments.
  • Wells were coated either with full-length sFRP-1 or with any one of a set of epitope-tagged sFRP-1 deletion mutants (Uren et al, JBiol Chem 275:4374-4382, 2000) or BSA control, and then sequentially incubated with soluble RANKL and reagents to detect RANKL bound to the wells.
  • the results shown are the mean +/- S.D. of triplicate measurements from a representative experiment.
  • FIG. 13 is a set of four graphs showing that binding of RANKL to bacterially expressed CRD in ELISA experiments is strong and may have two affinities.
  • FIG. 13A is a graph showing the binding of RANKL to wells coated with the CRD. Optical density in the wells is a measure of the amount of RANKL retained in the wells and is plotted as a function of the soluble RANKL concentration incubated in the wells.
  • FIG. 13B is a Scatchard plot of the RANKL binding data shown in FIG. 13 A. The binding appears to be characterized by more than one affinity.
  • FIG. 13C is a reformatting of the Scatchard analysis of FIG. 13B, pertaining to a putative higher-affinity binding site.
  • FIG. 13D is a reformatting of the Scatchard analysis of FIG. 13B, pertaining to a putative lower-affinity binding site.
  • FIG. 14 is a set of three graphs showing that the bacterially expressed CRD of sFRP-1 inhibits osteoclast formation in a variety of experimental models, including one that is not dependent on RANKL. These were: (1) RAW264.7 + TNF ⁇ + TGF ⁇ (FIG. 14A; Horwood et al, Journal of Immunology 166:4915-4921, 2001; Quinn et al, Journal of Bone and Mineral Research. 16, 1787- 1794, 2001 (2) the macrophage/monocyte cell line RAW264.7 + RANKL (FIG. 14B), and (3) bone marrow cells + RANKL + M-CSF (FIG. 14C). In each system, both RANKL-dependent (FIG. 14B and FIG.
  • FIG. 15 is a graph showing that sFRP-1 can inhibit osteoclast formation in RAW264.7 cells treated with a combination of cytokines that includes TNF ⁇ , but not RANKL.
  • the effect of sFRP-1 was assessed upon a RANKL-independent method of osteoclast formation using the monocyte /macrophage cell line RAW264.7 (Quinn et al, Journal of Bone and Mineral Research. 16, 1787- 1794, 2001) and was compared with that of osteoprotegerin.
  • FIG. 16 is a schematic diagram of one possible mechanism of sFRP-1 (SEQ ID NO:
  • sFRP-1 binding motif in the RANKL sequence is located just downstream from TACE cleavage sites (arrows).
  • TACE is the TNF ⁇ converting enzyme, which is known to process RANKL (L. Lum et al., J. Biol. Chem. 274: 13613-13618, 1999).
  • sFRP-1 binding to RANKL could alter the processing of RANKL by TACE, which in turn could alter RANKL activity.
  • FIG. 17 is a diagram showing one possible model of sFRP-l's role in osteoclast formation.
  • An osteoclast-supporting cell expressing RANKL interacts with sFRP-1 (SEQ ID NO: 3) resulting in the inhibition of osteoclast formation.
  • the peptide motif SEQ ID NO: 9 is added to the solution it binds to sFRP-1 (SEQ ID NO: 3) and promotes osteoclast differentiation.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 shows the cDNA sequence of human sFRP-1.
  • SEQ ID NO: 2 shows the nucleic acid sequence of the human sFRP-1 open reading frame.
  • SEQ TD NO: 3 shows the amino acid sequence of human sFRP-1.
  • SEQ ID NO: 4 shows the amino acid sequence of human sFRP-1-M/H.
  • SEQ YD NO: 5 shows the amino acid sequence of human sFRP- ⁇ l-M/H.
  • SEQ TD NO: 6 shows the amino acid sequence of human sFRP- ⁇ 2-M/H.
  • SEQ ID NO: 7 shows the amino acid sequence of human sFRP- ⁇ 3-M/H.
  • SEQ ID NO: 8 shows the amino acid sequence of human sFRP- ⁇ CRD-M H.
  • SEQ ID NO: 9 shows the amino acid sequence of the peptide motif.
  • SEQ ID NO: 10 shows the peptide motif from ANP receptor A (human).
  • SEQ ID NO: 11 shows the amino acid sequence of the A-E4 peptide.
  • SEQ ID NO: 12 shows the amino acid sequence of the A-F7 peptide.
  • SEQ ID NO: 13 shows the amino acid sequence of the netrin homology domain of sFRP-1.
  • SEQ ID NO: 14 shows the amino acid sequence of the A-C2 peptide.
  • SEQ ID NO: 15-26 show peptides generated for use in alanine scanning experiments.
  • SEQ ID NO: 27 shows the amino acid sequence of B-B9.
  • SEQ ID NO: 28 shows an amino acid sequence found in RANKL that contains a sequence similar to that of SEQ ID NO: 9.
  • SEQ ID NO: 29 shows an amino acid sequence found in a nerrin receptor that contains a sequence similar to that of SEQ ID NO: 9.
  • SEQ ID NOS: 30-39 show the nucleic acid sequences of various primers and probes used in PCR and hybridization experiments.
  • SEQ ID NO: 40 shows the amino acid sequence of the A-D9 peptide.
  • BSA bovine serum albumin
  • HSPG heparin-sulfate proteoglycan mAb: monoclonal antibody
  • MDCK Madin-Darby canine kidney
  • PBS phosphate-buffered saline sFRP : secreted Frizzled-related protein
  • Wnt Wnt proteins
  • Deviation from normal characteristics can be found in a control, a standard for a population, etc.
  • the abnormal condition is a disease condition, such as osteoporosis (characterized by a decrease in bone mass)
  • a few appropriate sources of normal characteristics might include an individual who is not suffering from the disease (e.g. osteoporosis), a population standard of individuals believed not to be suffering from the disease, etc.
  • abnormal can refer to a condition that is associated with a disease.
  • the term "associated with” includes an increased risk of developing the disease as well as the disease itself.
  • a certain abnormality such as a decrease in the expression of sFRP, which in turn upregulates osteoclast formation
  • the abnormality is predictive both of an increased risk of developing osteoporosis and of the presence of osteoporosis.
  • Abnormal protein expression refers to expression of a protein that is in some manner different from expression of the protein in a normal (wildtype) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues, such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein, compared to a control or standard amount; (5) expression of a decreased amount of the protein, compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); (8) alteration in post translational processing; and (9) alteration of the localized (e.g. organ or tissue specific)
  • Controls or standards appropriate for comparison to a sample, for the determination of abnormality include samples believed to be normal as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • CRD A cysteine rich domain that typically is about 120 amino acids in length and found on the amino terminal half of Fz proteins.
  • the CRD comprises sFRP-1 residues 38-166.
  • Met was added at the N-terminus to facilitate protein expression. Typically the Met is cleaved in the bacteria as the protein is processed.
  • the CRD sequence is shown below:
  • Detectable marker or label is any molecule or composition that is detectable by, for instance, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • labels including radioactive isotopes, enzyme substrates, co-factors, ligands, chermluminescent or fluorescent agents, haptens, enzymes, colloidal gold particles, colored latex particles, and epitope tags.
  • Epitope tags are short stretches of amino acids to which a specific antibody can be raised, which in some embodiments allows one to specifically identify and track the tagged protein that has been added to a living organism or to cultured cells. Detection of the tagged molecule can be achieved using a number of different techniques.
  • Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western"), and affinity chromatography.
  • Examples of useful epitope tags include FLAG, T7, HA (hemagglutinin) and myc.
  • Fluorophore A chemical compound, which when excited by exposure to a particular wavelength of light, emits light (i.e. fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or "color.” Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 ⁇ . Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-690 ⁇ .
  • fluorophores examples include for instance: 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l- sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- frifluoromethylcouluar (Cournaran 151); cyan
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodarnine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); NNN',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.
  • ROX 6-carboxy-X-rhodamine
  • fluorophores include GFP (green fluorescent protein), LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivatives thereof.
  • GFP green fluorescent protein
  • LissamineTM diethylaminocoumarin
  • fluorescein chlorotriazinyl diethylaminocoumarin
  • fluorescein chlorotriazinyl 1,4-dichlororhodamine
  • xanthene 1,7-dichlororhodamine
  • Fusion protein A protein comprising two amino acid sequences that are not found joined together in nature.
  • the term "sFRP peptide motif fusion protein” refers to a protein that comprises a first amino acid sequence that binds sFRP and a second amino acid sequence.
  • the sFRP binding motif and the second amino acid sequence may alternatively be referred to as domains of the fusion protein.
  • the present disclosure provides fusion proteins comprising first and second domains, wherein the first domain includes a peptide motif that binds sFRP.
  • the link between the first and second domains of the fusion protein is typically, but not necessarily, a peptide linkage.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs (i.e. other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles).
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Linker group or linking group is a "chemical arm" between a protein or peptide and a detectable marker.
  • each of the reactants must contain the necessary groups to link the peptide to the detectable marker.
  • Representative combinations of such groups are amino with carboxyl to form amide linkages, or carboxy with hydroxy to form ester linkages or amino with alkyl halides to form alkylarnino linkages, or thiols with thiols to form disulfides, or thiols with maleimides or alkylhalides to form thioethers. Hydroxyl, carboxyl, amino and other functionalities, where not present may be introduced by known methods.
  • linking groups may be employed.
  • the structure of the linkage should be a stable covalent linkage formed to attach the protein or peptide to the detectable marker or label.
  • the linking group may be designed to be either hydrophilic or hydrophobic in order to enhance the desired binding characteristics of the ligand and the receptor.
  • the covalent linkages should be stable relative to the solution conditions under which the ligand and linking group are subjected.
  • preferred linking groups will be from 1-20 carbons and 0-10 heteroatoms (NH, O, S) and may be branched or straight chain. Without limiting the foregoing, it should be obvious to one skilled in the art that only combinations of atoms that are chemically compatible comprise the linking group.
  • Mimetic A molecule (such as an organic chemical compound) that mimics the activity of a protein, such as sFRP or its fragments, the peptide motif (such as SEQ ID NO: 9 or SEQ ID NO: 40), or variants or fusions thereof.
  • Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific inhibitory activity or agonist activity.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs," in Klegerman & Groves, eds., Pharmaceutical Biotechnology, Interpharm Press:
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length. In several embodiments an oligonucleotide is at least 10, 20, 30, 40, or 50 nucleotides in length.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • Osteoclasts are large, multinucleate cells that actively reabsorb bone. Osteoclasts are derived from hematopoietic stem cells and share phenotypic characteristics with circulating monocytes and tissue macrophages. They are formed from a population of the circulating mononuclear cells that are recruited from the blood to the bone surface where they undergo differentiation and fusion to form multinucleated cells.
  • Osteopetrosis is a family of diseases characterized by the failure of the long bones to be remodeled. The resulting long bones have cartilagenous infiltration towards the center of the bone from the growth plate and a poorly remodeled center. While osteoporosis can be caused by too many osteoclasts, osteopetrosis can be caused by not having sufficient numbers of these cells.
  • Osteoclasts are commonly found in degenerative bone diseases at sites of osteolysis. Osteoclast overproduction is associated with diseases such as hype arathyroidism and Paget's disease. Osteoclasts are also seen at sites of inflammatory reactions associated with aseptic loosening of total hip prosthesis, rheumatoid arthritis, and periodontitis. Two cytokines produced by inflammatory cells that may have direct effects on osteoclast formation and function are interleukin-1 (IL-1) and tumor necrosis factor (TNF- ⁇ ).
  • IL-1 interleukin-1
  • TNF- ⁇ tumor necrosis factor
  • Peptide motif An amino acid sequence that binds sFRP-1.
  • a peptide motif is sequence of two or more peptide-linked amino acids that provides a characteristic structure and or function.
  • a peptide motif can be found in more than one protein or more than once in a single protein.
  • the peptide motif shown in SEQ ID NO: 9 is characterized by its ability to bind to sFRP and modulate sFRP activity.
  • the three core residues of SEQ ID NO: 9 D-G-R
  • a peptide motif includes these three amino acids.
  • a peptide motif in another embodiment includes the five core amino acids of SEQ ID NO: 9 (V-D-G-R-W).
  • SEQ ID NOS: 9-11, 14-17, and 24-26 motifs that bind to sFRP and can be capable of modulating sFRP activity.
  • amino acid sequence of one embodiment of the peptide motif that binds sFRP-1 is shown in SEQ ID NO: 9, one of skill in the art will appreciate that variations in this amino acid sequence, such as 1, 2, or 3 deletions, additions, or substitutions, can be made without substantially affecting the activities of the peptide motif.
  • the term "peptide motif encompasses both the motif provided in SEQ ID NO: 9, and the additional peptide motifs provided in SEQ ID NOS: 10 and 11 and 14-26, as well as amino acid sequences that are based on these sequences but which include one or more sequence variants and fragments of these sequences that contain at least 3, 4, 5, or 6 contiguous amino acids of the peptide motif.
  • sequence variants or fragments can also be defined in the degree of amino acid sequence identity that they share with the amino acid sequence shown in SEQ ID NO: 9.
  • peptide motif sequence variants will share at least 80% sequence identity with the sequences shown in SEQ ED NOS: 9- 12 and 14-26. More highly conserved variants will share at least 90%, at least 95%, or at least 98% sequence identity with the sequences shown in SEQ ID NOS: 9-12, 14-17, and 24-26.
  • the peptide motif is characterized by its ability to bind to sFRP. This activity can be tested using the ELISA assay described below in the methods section.
  • the peptide motifs ability to bind to sFRP and modulate sFRP activity is beneficial in a number of applications, including clinical applications such as in the treatment of diseases associated with abnormal bone remodeling, and more specifically when increased osteoclast activity is desired.
  • Peptide tag A peptide sequence that is attached (for instance through genetic engineering) to another peptide or a protein, to provide a function to the resultant fusion.
  • Peptide tags are usually relatively short in comparison to a protein to which they are fused; by way of example, peptide tags are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids.
  • peptide tags usually are four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids.
  • a peptide tag will be no more than about 100 amino acids in length, and may be no more than about 75, no more than about 50, no more than about 40, or no more than about 30.
  • Peptide tags confer one or more different functions to a fusion protein (thereby "functionalizing" that protein), and such functions can include antibody binding (an epitope tag), purification, and differentiation (e.g., from a native protein).
  • an epitope tag an epitope tag
  • purification e.g., from a native protein
  • differentiation e.g., from a native protein
  • a recognition site for a protease, for which a binding antibody is known can be used as a specifically cleavable epitope tag.
  • the use of such a cleavable tag can provide selective cleavage and activation of a protein (e.g., by replacing the cleavage site in TGF- ⁇ l with that for pro-caspase 3.
  • Detection of the tagged molecule ca be achieved using a number of different techniques. These include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western”), and affinity chromatography.
  • Epitope tags add a known epitope (antibody binding site) on the subject protein, providing binding of a known and often high-affinity antibody, and thereby allowing one to specifically identify and track the tagged protein that has been added to a living organism or to cultured cells.
  • epitope tags include the myc, T7, GST, GFP, HA (hemagglutinin) and FLAG tags. The first four examples are epitopes derived from existing molecules.
  • FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Patent Nos. 4,703,004 and 4,851,341).
  • Purification tags are used to permit easy purification of the tagged protein, such as by affinity chromatography.
  • a well-known purification tag is the hexa-histidine (6x His) tag, literally a sequence of six histidine residues.
  • the 6x His protein purification system is available commercially from QIAGEN (Valencia, CA), under the name of QlAexpress®.
  • a single tag peptide can serve more than one purpose; any attached tag, for instance, will increase the molecular weight of the fusion protein and thereby permit differentiation between the tagged and native proteins.
  • Antibodies specific for an "epitope tag” can be used to construct an immunoaffinity column, thus permitting an epitope tag to be used for purification of the tagged protein.
  • monoclonal antibodies specific for a purification tag are available (e.g. anti-6x His peptide monoclonal antibodies, which are available through QIAGEN or CLONTECH, Palo Alto, CA).
  • compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed are conventional. Remingto 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g. powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide A nucleic acid sequence including at least two nucleic acid residues.
  • Polypeptide A protein fragment including at least two amino acid residues.
  • Protein Fragment An amino acid sequence that contains fewer amino acid residues than are found in a naturally occurring protein and including at least two amino acid residues. For example, if a naturally occurring protein, i.e. a protein expressed from a gene, is 300 amino acid residues long, a polypeptide derived from the protein could have 299 amino acid residues or less. In particular examples, the polypeptide could have less than 200, 175, 150, 125, 100, 75, 50, or 25 amino acid residues.
  • Purified The term purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified protein or peptide preparation is one in which the protein or peptide is more pure than the protein or peptide in its natural environment within a cell.
  • Such proteins or peptides may be produced, for example, by standard purification techniques, or by recombinant expression.
  • a preparation of a protein or peptide is purified such that the protein or peptide represents at least 50%, for example, or at least 70%, of the total protein content of the preparation.
  • RANK and RANKL The receptor activator of NF- ⁇ B (RANK) is a member of the tumor necrosis factor (TNF) receptor superfamily.
  • TNF tumor necrosis factor
  • the ligand, receptor activator of NF-KB ligand (RANKL) is a member of the TNF superfamily, and has been characterized in multiple settings and variously termed Osteoclast Differentiation Factor (ODF), Tumor Necrosis Factor-Related
  • RANK is a Type I transmembrane protein having 616 amino acid residues that interacts with TTSfF-receptor associated factor 3 (TRAF3).
  • TTSfF-receptor associated factor 3 TTSfF-receptor associated factor 3
  • RANKL membrane bound RANK ligand
  • NF- ⁇ ubiquitous transcription factor that is most extensively utilized in cells of the immune system
  • RANK is expressed on osteoclast precursors and mature osteoclasts.
  • RANKL produced by osteoblasts stimulates the formation and activity of osteoclasts, which facilitates normal bone development and remodeling.
  • Gene targeting of either RANKL or RANK results in osteopetrosis (increased bone mass), as well as severe defects in lymph node formation.
  • Osteoprotegerin (OPG) is a soluble factor that also belongs to the TNF receptor family. OPG binds to RANKL, and inhibits the formation of functional multinucleate osteoclasts in vitro.
  • OPG binds to RANKL, and inhibits the formation of functional multinucleate osteoclasts in vitro.
  • Overexpression of OPG in transgenic mice causes severe osteopetrosis, with a loss of marrow cavities and profound depletion of osteoclasts. The same effects were observed upon administration of OPG in normal mice.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g. by genetic engineering techniques.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of sFRP (the prototypical member of which is shown in SEQ ID NO: 1), or the peptide motif that binds sFRP (for example SEQ ID NO:9), disclosed herein, will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al, J. Mol. Biol 215:403- 410, 1990) is available from several sources, including the National Center for Biotechnology Jjformation (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • Variants of sFRP, sFRP fragments, or the peptide motif that binds sFRP are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of sFRP, sFRP fragments or the peptide motif (for example SEQ ID NO: 9) using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default
  • BLOSUM62 matrix set to default parameters (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, or 98% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85% or at least 90%, 95%, or 98% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Maryland. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • sFRP Secreted Frizzled-related protein
  • sFRP is a secreted protein that consists of approximately 300 amino acids, including a CRD that is typically between 30% and 50% identical to the (cysteine-rich domain) CRD of the Fz protein family members.
  • CRD Cysteine-rich domain
  • SEQ ID NO: 1 The nucleic acid and amino acid sequences of other members of the sFRP family can be found at the National Center for Biotechnology Website, for example GenBank Accession No.
  • AF218056 (Gallus gallus FRP-2), GenBank Accession No AV354083 (Mus musculus- ⁇ HP-1), GenBank Accession No AV304328 (Mus musculus s-FRP-2), GenBank Accession No U24163 (homo sapiens sFRP-3/FrzB) and GenBank Accession No AI587049 (Homo sapiens sFRP-1).
  • the open reading frame of the prototypical sFRP is shown in SEQ ID NO: 2, while the sequence of the protein is shown in SEQ ID NO: 3.
  • sFRP binds to RANKL and inhibits osteoclast formation.
  • sFRP-1 binding activity and its ability to modulate osteoclast formation can be assayed using the ELISA and osteoclastogenesis bioassay methods described herein.
  • the ability of sFRP-1 protein, or a fragment thereof, to perform these activities is beneficial in a number of applications, including clinical applications such as in the treatment of diseases associated with abnormal bone remodeling.
  • amino acid sequence of the prototypical sFRP is shown in SEQ ID NO: 3, one of skill in the art will appreciate that variations in this amino acid sequence, such as 1, 2, 5, 10, 20, 30, 40, or 50, deletions, additions, or substitutions (including conservative amino acid substitutions), can be made without substantially affecting the activities of the protein (or fragments of the protein) discussed above.
  • the term "sFRP" fragments encompasses both the proteins having the amino acid sequences shown in SEQ ID NOs: 4-8, as well as amino acid sequences that are based on these sequences but which include one or more sequence variants. Such sequence variants can also be defined in the degree of amino acid sequence identity that they share with the amino acid sequence shown in SEQ ID NOs: 4-8.
  • sFRP sequence variants will share at least 80% sequence identity with the sequences shown in SEQ ID NOs: 4-8. More higlily conserved variants will share at least 90%, at least 95%, or at least 98% sequence identity with the sequences shown in SEQ ID NOs: 4-8. In addition to sharing sequence identity with the prototypical sFRP protein sequence, such sequence variants possess the ability to bind to TNF family members such as RANKL.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • Therapeutically effective dose A dose sufficient to prevent advancement, or to cause regression of the disease, or which is capable of relieving symptoms caused by the disease.
  • TNF family of proteins The Tumor Necrosis (TNF) family of proteins contains both membrane bound ligands and soluble proteins. Some family members, such as TNF and RANKL, are active in both membrane-anchored and soluble forms, the latter being enzymatically released into solution, notably by TACE (TNF alpha converting enzyme) (J. Hardy, Proc. Natl. Acad. Sci. U.S.A. 94:2095-2097, 1997; J.D. Buxbaum et al, Proc. Natl. Acad. Sci. U.S.A. 89: 10075-10078, 1992).
  • TACE TNF alpha converting enzyme
  • TNF family members The primary area of homology among TNF family members is a stretch of 150 amino acid residues in the carboxy-terminus that is situated in the extracellular space. This domain is responsible for binding to cognate members of the TNF receptor family.
  • This family of receptor proteins is characterized by four domains with regularly spaced cysteine residues: each has a single transmembrane domain and binds either TNF ⁇ or TNF ⁇ .
  • Members of the family include, for example, TNFRI, TNFRII, Fas, CD30, and CD30.
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • a vector can also include a sequence encoding an amino acid motif that facilitates the isolation of the desired protein product such as a sequence encoding maltose binding protein, c-myc, or GST.
  • WNT One group of genes and the proteins encoded by them that play an important role in regulating cellular development is the Wnt family of glycoproteins.
  • Wnt proteins are a family of growth factors consisting of more than a dozen structurally related molecules and are involved in the regulation of fundamental biological processes, like apoptosis, embryogenesis, organogenesis, morphogenesis and turnorigenesis. These polypeptides are multipotent factors and have similar biological activities to other secretory proteins like fransfo ⁇ riing growth factor (TGF)- ⁇ , fibroblast growth factors (FGFs), nerve growth factor (NGF), and bone morphogenetic proteins (BMPs).
  • TGF fransfo ⁇ riing growth factor
  • FGFs fibroblast growth factors
  • NGF nerve growth factor
  • BMPs bone morphogenetic proteins
  • a member of the Wnt growth factor family is preferentially expressed in bone tissue and in bone-derived cells, and appears to be involved in maintaining the mature osteoblast (bone-forming cell) phenotype.
  • a peptide motif has been demonstrated to bind to sFRP-1 (SEQ ID NO: 3) and inhibit the ability of sFRP-1 to down regulate the formation of osteoclasts.
  • the peptide motif has the formula:
  • RI is selected from the group consisting of (a) Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr (AGT), (c) Gln-Ala-Thr (QAT), and (d) Gln-Gly-Ala (QGA).
  • R2 is selected from the group Leu, Val, Ala (L, V, A) or a conservative substitution therefor.
  • R8 is selected from the group consisting of Leu or Val (L or V) or a conservative substitution therefor.
  • R9 is selected from the group consisting of (a) Gin (Q), (b) Gln-Gly-Glu (QGE), (c) Gin-Leu (QL), (d) Ala-Leu (AL), (e) Gin-Ala (QA) and (f) Thr-Asn-Pro- His-His (TNPHH).
  • R3 is V
  • R4 is D
  • R5 is R
  • R6 is G
  • R7 is W.
  • RI is selected from the group consisting of (a) Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr (AGT), (c) Gln-Ala-Thr (QAT), and (d) Gln-Gly-Ala (QGA);
  • R2 is selected from the group Leu, Val, Ala (L, V, A) or a conservative substitution therefor;
  • R8 is selected from the group consisting of Leu or Val (L or V) or a conservative substitution therefor ;
  • R9 is selected from the group consisting of (a)Gln (Q), (b) Gln-Gly-Glu (QGE), (c)GIn-Leu (QL), (d) Ala-Leu (AL), (e) Gin-Ala (QA) and (f) Thr-Asn-Pro- His-
  • R3 is V
  • R4 is D
  • R5 is R
  • R6 is G
  • R7 is W
  • RI is selected from the group consisting of (a) Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr (AGT), (c) Gln-Ala-Thr (QAT), and (d) Gln-Gly-Ala (QGA).
  • QGT Gln-Gly-Thr
  • AAT Ala-Gly-Thr
  • QGA Gln-Ala-Thr
  • R3 is V
  • R4 is D
  • R5 is R
  • R6 is G
  • R7 is W
  • R2 is selected from the group Leu, Val, Ala (L, V, A) or conservative substitutions therefor.
  • R3 is V, R4 is D, R5 is R, R6 is G, and R7 is W and R8 is selected from the group consisting of Leu, Val, Ala (L, V, A) and conservative substitutions therefor.
  • R3 is V, R4 is D, R5 is R, R6 is G, and R7 is W and R9 is selected from the group consisting of (a)Gln (Q), (b)Gln-Gly-Glu (QGE), (c)Gln-Leu (QL), (d) Ala-Leu (AL), (e) Gin-Ala (QA) and (f) Thr-Asn-Pro- His-His (TNPHH).
  • a peptide motif is SEQ ID NO: 9.
  • the sFRP binding peptide is less than 30 amino acids in length.
  • the peptide is less than 20 amino acids in length.
  • the peptide is less than 10 amino acids in length.
  • a peptide motif that binds sFRP has also allowed other proteins to be identified, which are characterized by the presence of a sequence resembling the peptide motif in their amino acid sequences, and by their ability to bind to sFRP family members. These peptides interfere with sFRP activity, for example binding RANKL or a TNF family member, or osteoclastogenesis stimulating activity. Accordingly, the disclosure provides methods of controlling bone remodeling.
  • the peptide motif disclosed herein can be used to bind to sFRP-1 and effectively upregulate osteoclast differentiation.
  • Increased osteoclast production is desirable for the treatment of disorders where there is too much bone formation (for example, achondroplasia, Albright's osteodystrophy, and osteopetrosis).
  • the disclosure also provides methods of providing sFRP to increase bone mass (see FIG. 17).
  • An increase in bone mass is desirable for the treatment of disorders such as postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, hyperparathyroidism, rheumatoid arthritis, periodontitis, and hypercalcemia of malignancy.
  • Some embodiments of the disclosure provide isolated polypeptides, including the amino acid sequence shown in SEQ ID NO: 9; conservative amino acid substitutions of the amino acid sequence shown in SEQ ID NO: 9; and amino acid sequences that share at least 80% sequence identity with the sequence shown in SEQ ED NO: 9.
  • These polypeptides are capable of binding sFRP-1 (SEQ ID NO: 3) and interfering with sFRP activity, for example osteoclastogenesis activity. Examples of such polypeptides are provided in SEQ ID NOS: 10-12 and 14-29.
  • the disclosure also provides nucleic acid sequences that encode the peptide motif that binds sFRP and the variants of the peptide motif that binds sFRP that are described in the paragraph above. These nucleic acid sequences can be placed in vectors, and the vectors can be used to transform host cells. The transformed host cells are subsequently useful for, among other things, producing the above-described polypeptides.
  • the disclosure provides methods of enhancing osteoclast differentiation in a subject. These methods include providing an effective amount of a peptide that includes the motif (such as SEQ ID NO: 9), or variants, or fragments thereof to increase osteoclast differentiation. Such methods are useful for treating subjects suspected of having abnormal bone remodeling (e.g. achondroplasia, Albright's osteodystrophy, or osteopetrosis).
  • achondroplasia e.g. achondroplasia, Albright's osteodystrophy, or osteopetrosis.
  • the disclosure also provides methods of inhibiting osteoclast formation in a subject. These methods include administering sFRP-1 (SEQ ID NO: 3), variants of sFRP-1 (SEQ ID NO: 3), or fusions, or fragments of sFRP-1 (SEQ ID NO: 3). Administering these peptides includes administration and expression of nucleic acids that encode the peptides.
  • the administered proteins or peptides are characterized by their ability to bind to RANKL, for example, human RANKL termed "TRANCE" (AF013171), human RANKL (AF019047), and human RANKL termed "OPGL” (AF053712) and inhibit osteoclast formation.
  • osteoclast formation will be useful for treating osteopathic disorders such as postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, hyperparathyroidism, rheumatoid arthritis, periodontitis, and hypercalcemia of malignancy.
  • osteopathic disorders such as postmenopausal osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, hyperparathyroidism, rheumatoid arthritis, periodontitis, and hypercalcemia of malignancy.
  • the peptide motif that binds sFRP, and fragments and variants thereof, are also useful for modulating T-cell activity. Accordingly, the disclosure provides methods of modulating T-cell activity. These methods include providing an effective amount of the peptide motif that binds sFRP (such as SEQ ID NO:9), or fragments and variants thereof, sufficient to change T-cell interaction with dendritic cells or osteoclast progenitor cells. Examples of changes in the interaction between the T-cell and the dendritic cell include an increase in dendritic cell survival, and T cell proliferation, in a mixed lymphocyte reaction, as described for RANKL/RANK signaling (D.M. Anderson et al,
  • Modulating T-cell activity is desirable in subjects suspected of having, for example, toxic shock, sepsis, grafit- versus-host reactions, or acute inflammatory reactions.
  • the disclosure also provides methods of screening for sFRP proteins, and fragments, and variants thereof, that bind to members of the TNF family of proteins. These methods include contacting an sFRP protein with at least one TNF family member, and detecting TNF family member binding to the sFRP protein.
  • TNF family members include RANKL, Apo2/TRAIL, FasL, CD40L, CD27L, CD30L, Apo3L/TWEAK, TNF and LT-alpha (S.J. Baker and E.P. Reddy, Oncogene 17: 3261-3270, 1998).
  • sFRP-1 SEQ ID NO: 3
  • sFRP-2 GenBank Accession No. MMU88567, incorporated herein by reference
  • sFRP-3 GenBank Accession No. MMU88568, incorporated herein by reference
  • sFRP-4 GenBank Accession No. AF012891, incorporated herein by reference
  • sFRP-5 GenBank Accession No. AF117758, incorporated herein by reference.
  • the disclosure also provides the purified peptide shown in SEQ ID NO: 14. This peptide is useful for stimulating osteoclast differentiation in vitro and in vivo. When the peptide is used in vivo it can be administered to subjects to increase osteoclast differentiation.
  • sFRP, fragments, fusions, and variants thereof, as well as the peptide motif sFRP fragments and variants thereof can be purified from MDCK cells (ATCC NO. CCL-34) transfected with sFRP encoding vectors as described below.
  • sFRP fragments and variants thereof can also be purified from a tissue source using conventional biochemical techniques, or produced recombinantly in either prokaryotic or eukaryotic cells using methods well-known in the art (for example, those described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989). The recombinant expression of sFRP fragments is described in (Uren et al, J. Biol. Chem. 275:4374-4382,
  • nucleic acid sequences encoding sFRP family members are available on GenBank, and include the cDNA sequence shown in SEQ ID NO: 1.
  • Recombinant sFRP fragments, fusions, and variants thereof, as well as the binding motif (SEQ ID NO: 9) and variants thereof, can be obtained using commercial systems designed for optimal expression and purification of fusion proteins.
  • fusion proteins typically include a protein tag that facilitates purification.
  • Examples of such systems include: the pMAL protein fusion and purification system (New England Biolabs, Inc., Beverly, MA); the GST gene fusion system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ); and the pTrcHis expression vector system (Invitrogen, Carlsbad, CA).
  • the pMAL expression system utilizes a vector that adds a maltose binding protein to the expressed protein.
  • the fusion protein is expressed in E. coli., and the fusion protein is purified from a crude cell extract using an amylose column. If necessary, the maltose binding protein domain can be cleaved from the fusion protein by treatment with a suitable protease, such as Factor Xa. The maltose-binding fragment can then be removed from the preparation by passage over a second amylose column.
  • Eukaryotic expression systems can also be employed, including Pichia, tobacco and Baculovirus expression systems, such as those available commercially from Invitrogen.
  • the entire sFRP protein, variants and fragments thereof or the peptide binding motif can be produced by ligating the open reading frame (ORF) of the desired sequence into the vector.
  • ORF open reading frame
  • the ORF must be operably linked to the vector, i.e. must be joined such that the reading frame of the ORF is aligned with the reading frame of the protein tag.
  • an ORF encoding the desired fragment can be amplified by polymerase chain reaction (PCR) from the sFRP cDNA, cloned, purified and then ligated into the expression vector. Alternatively, the amplified fragment can be ligated directly into the expression vector. It can also be possible, depending on the availability of suitable restriction sites in the sFRP cDNA, to obtain the desired fragment by appropriate restriction endonuclease digestion, such that it can be directly cloned into the expression vector.
  • PCR polymerase chain reaction
  • Purification of the expressed protein can be achieved either using the purification regimen appropriate for the expression tag (if a commercial expression purification system is used), or conventional affinity chromatography using antibodies, preferably monoclonal antibodies, that recognize the appropriate regions of sFRP can be employed or chromatography procedures established for sFRPs.
  • fragments or protein fragments containing the peptide motif for example, SEQ ID NO: 9
  • such fragments alternatively canbe generated through digestion of a full-length protein with various proteases. The fragments can then be separated based on their unique size, charge or other characteristics. Such fragments can also be synthetically generated through the use of known peptide synthesis methods.
  • Osteoclasts are large, multinucleate cells that actively reabsorb bone, are derived from hematopoietic stem cells, and share phenotypic characteristics with circulating monocytes and tissue macrophages. They are formed from a population of the circulating mononuclear cells that are recruited from the blood to the bone surface, where they undergo differentiation and fusion to form multinucleated cells.
  • Osteopetrosis is a family of diseases characterized by the failure of the long bones to be remodeled. The resulting long bones have cartilagenous infiltration towards the center of the bone from the growth plate and a poorly remodeled center. While osteoporosis can be caused by osteoclasts that are too numerous or too active, osteopetrosis can be caused by not having sufficient numbers of these cells, or by their inadequate activity. Thus, enhancement of osteoclast differentiation is desirable in subjects with abnormal bone remodeling, such as achondroplasia and osteopetrosis. Methods of administration of these FRP-1 to inhibit osteoclast differentiation in a subject are described below.
  • sFRP-1 can be used to inhibit osteoclast formation.
  • Loss of ovarian function following menopause often results in a progressive loss of trabecular bone mass and eventually to osteoporosis. This bone loss is in part due to the increased production of osteoclasts.
  • This increased production of osteoclasts appears to be due to the increased elaboration by support cells of osteoclastogenic cytokines such as IL-1, tumor necrosis factor, and IL-6, all of which are negatively regulated by estrogens.
  • Osteoclasts are also implicated in degenerative bone diseases at sites of osteolysis. Likewise, osteoclast overproduction is associated with diseases such as hype ⁇ arathyroidism and Paget's disease. Osteoclasts are also seen at sites of inflammatory reactions associated with aseptic loosening of total hip prosthesis, rheumatoid arthritis, and periodontitis. Two cytokines produced by inflammatory cells that may have direct effects on osteoclast formation and function are interleukin-1 (IL-1) and tumor necrosis factor (TNF- ⁇ ). Thus, inhibition of osteoclast formation is desirable in subjects with bone disorders characterized by unwanted bone resorption.
  • IL-1 interleukin-1
  • TNF- ⁇ tumor necrosis factor
  • sFRP- 1 can have clinical utility in conditions where excessive osteoclast activity has pathological consequences.
  • Osteoporosis and rheumatoid arthritis are examples of conditions that are particularly good targets for sFRP-1 therapy because soluble RANKL from T cells is thought to have an important role in the bone loss associated with these diseases.
  • Methods of administration of sFRP-1 to inhibit osteoclast formation in a subject are disclosed herein.
  • disorders of calcium homeostasis can also be affected by osteoclast activity.
  • osteoclasts are able to mobilize calcium from bone to affect hypocalcemic states.
  • inhibition of osteoclasts can help nnnimize mobilization of in hypercalcemic states.
  • modulation of osteoclast activity can be used as a therapeutic intervention to treat hypocalcemia and hypercalcemia.
  • the sFRP-binding peptides described herein are effective for treatment of conditions or diseases that involve the immune system, for instance conditions (including clinical treatments) that inhibit (or suppress) the immune system.
  • conditions or diseases that involve the immune system
  • conditions including clinical treatments
  • immunomodulatory compounds General information about the therapeutic use of immunomodulatory compounds is well known, and can be found for instance in U.S. Patent Nos. 5,632,983; 5,726,156; and 5,861,483.
  • the peptide motif disclosed herein is of use in modulating antigen presentation.
  • T-cells produce RANKL, and dendritic cells express RANK.
  • T cells can be exposed to a polypeptide including the peptide motif, for example administration of the polypeptide to a subject.
  • the administration of the polypeptide results in an increase in RANKL, and subsequently the binding of RANK to RANKL on T cells.
  • administration of a polypeptide including the sFRP binding peptide disclosed herein results in increased antigen presentation, and a corresponding upregulation of an immune response against the antigen.
  • Immune deficiencies e.g., deficiencies of one or more type of immune cells, or of one or more immunological factors
  • immune suppressive medical treatment acute and/or chronic infection, and aging
  • aging can be treated using the methods and compositions described herein.
  • a general overview of immunosuppressive conditions and diseases can be found in Harrisons "Principles of Internal Medicine," 14 th Edition, McGraw-Hill, 1998, and particularly in chapter 86 (Principles of Cancer Therapy), chapter 88 (Melanoma and other Skin Cancers), chapter 307 (Primary Immune Deficiency Diseases), and chapter 308 (Human Immunodeficiency Virus Diseases).
  • a polypeptide including the sFRP binding peptide motif is administered to an immunosuppressed subject, such as a subject receiving immunosuppressive medical treatment, a subject with an age-linked immunodeficiency, or a subject that is infected with a human immunodeficiency virus.
  • an immunosuppressed subject such as a subject receiving immunosuppressive medical treatment, a subject with an age-linked immunodeficiency, or a subject that is infected with a human immunodeficiency virus.
  • the peptides disclosed herein are utilized to activate the immune system against various diseases, both chronic and acute.
  • Subject infections include bacterial and viral infections, as well as infestations caused by eukaryotic pathogens and parasites. More particularly, immunostimulatory sFRP- 1 -binding peptide-treatment can be used in the treatment of HIV disease.
  • compositions containing the peptide motif also have utility in disrupting the interaction of sFRP-1 with other proteins.
  • sFRP-1 binding to the ANP receptor A can regulate the release of sodium and fluid in the kidney and eye. It has been demonstrated that the relevant components of natriuretic peptide system are functionally expressed in the human eye where they are believed to serve as modulators of intraocular pressure (J. Ortego and M. Coca-Prados, Biochem. Biophys. Res. Commun. 258: 21-28, 1999).
  • sFRP- 1 or its binding peptide can have an important impact on the release of fluid into the eye with resultant changes in the intraocular pressure.
  • a polypeptide that includes a peptide motif that binds sFRP such as a polypeptide including SEQ ID NO:9, is administered to a subject to decrease intraocular pressure.
  • the polypeptide including the peptide motif that binds sFRP is administered to decrease intraocular pressure in a subject with glaucoma (see Johnson and R.C. Tschumper, Invest. Ophthahnol. Vis. Sci. 28: 945-953, 1987).
  • the peptide can be administered intraocularly (for example in a sustained release intraocular implant).
  • the polypeptide may be a ⁇ 'ministered systemically, in a therapeutically effective amount sufficient to inhibit production of aqueous humor in the anterior chamber of the eye.
  • the peptide motif that binds sFRP can be used in screening for the identification of proteins and other compounds that bind to, or otherwise directly interact with sFRP or fragments thereof, such as a mimetic.
  • the proteins include members of the TNF family of proteins such as, RANKL, TRAIL, FasL, CD40L, CD27L, CD30L, and NGF.
  • a cell lysate or tissue homogenate can be screened for proteins or other compounds that disrupt sFRP/TNF or sFRP/peptide motif binding.
  • any of a variety of exogenous compounds, both naturally occurring and/or synthetic e.g.
  • libraries of small molecules or peptides can be screened for the ability to disrupt sFRP/TNF or sFRP/peptide motif binding (such as the ability to disrupt binding of a peptide having a sequence as set forth as SEQ ID NO: 9 with TNF or RANKL).
  • Small molecules are particularly preferred in this context because they are more readily absorbed after oral administration, have fewer potential antigenic determinants, and/or are more likely to cross the blood brain barrier than larger molecules such as nucleic acids or proteins.
  • deletion mutants i.e. the fragments of sFRP shown in the sequence listing
  • a polypeptide including a peptide motif that binds sFRP can serve as a "lead compound.”
  • sequential modification of small molecules e.g. amino acid residue replacement with peptides; functional group replacement with peptide or non-peptide compounds
  • Such development generally proceeds from a "lead compound” which is shown to have at least some of the activity (e.g. modulates osteoclastogenesis) of the desired pharmaceutical.
  • the activity e.g. modulates osteoclastogenesis
  • structural comparison of the molecules can greatly inform the skilled practitioner by suggesting portions of the lead compounds that should be conserved, and portions that can be varied in the design of new candidate compounds.
  • the present disclosure also provides potential lead compounds as well as means of identifying such lead compounds that can be modified sequentially to produce new candidate compounds for use in the treatment of diseases associated with abnormal osteoclast activity, i.e. arthritis.
  • agents that disrupt sFRP/peptide motif binding can be monitored using the osteoclast differentiation assays described below.
  • Agents that disrupt sFRP binding and enhance osteoclastogenesis are useful for treating conditions associated with increased bone mass and agents that are found to enhance sFRP/TNF binding are useful for treating diseases associated with decreased bone mass (e.g. see FIG. 17).
  • Methods of detecting such binding include the ELISA assays described below, as well as other methods that involve monitoring changes in fluorescence, molecular weight, or the concentration of either sFRP, or proteins containing the peptide motif that binds sFRP either in a soluble phase or in a substrate-bound phase.
  • the peptide motif has a sequence as set forth as SEQ ID NO:9.
  • the candidate compounds can then be produced in quantities sufficient for pharmaceutical administration or testing (e.g. ⁇ g or mg or greater quantities), and formulated in a pharmaceutically acceptable carrier (see, e.g. Remington's Pharmaceutical Sciences, Gennaro, A., ed., Mack Pub., 1990). These candidate compounds can then be administered to the transformed cells of the disclosure, to the transgenic animal models of the disclosure, to cell lines derived from the animal models or from human patients.
  • proteins or other compounds identified by these methods can be purified and characterized by any of the standard methods known in the art. Proteins can, for example, be purified and separated using elecrrophoretic (e.g. SDS-PAGE, 2D PAGE) or chromatographic (e.g. HPLC) techniques and can then be microsequenced. For proteins with a blocked N-terminus, cleavage (e.g. by CNBr and/or trypsin) of the particular binding protein is used to release peptide fragments. Further purification/characterization by elecrrophoretic (e.g. SDS-PAGE, 2D PAGE) or chromatographic (e.g. HPLC) techniques and can then be microsequenced. For proteins with a blocked N-terminus, cleavage (e.g. by CNBr and/or trypsin) of the particular binding protein is used to release peptide fragments. Further purification/characterization by
  • HPLC and microsequencing and/or mass spectrometry by conventional methods provides internal sequence data on such blocked proteins.
  • standard organic chemical analysis techniques e.g. IR, NMR and mass spectrometry; functional group analysis; X-ray crystallography
  • Methods for screening cellular lysates, tissue homogenates, or small molecule libraries for candidate sFRP disrupting molecules are well known in the art and, in light of the present disclosure, can now be employed to identify compounds which disrupt sFRP binding to the peptide motif (for example SEQ ID NO:9) or TNF family members such as RANKL or TRAIL.
  • sFRP protein-binding protein
  • a fragment thereof or the peptide motif for example a fragment of about three or about five amino acids of SEQ ID NO: 9
  • a substrate e.g. a column or filter
  • a solution containing a TNF receptor or a sFRP family member protein can be introduced to the column to allow formation of the sFRP/TNF or peptide motif/sFRP complex.
  • a solution including the test compound(s) is introduced to the column under conditions that are permissive for binding.
  • test compounds can be immobilized and a solution containing sFRP/RANKL or sFRP/peptide motif (for example SEQ ID NO: 9) can be contacted with the column, filter or other substrate.
  • a solution containing sFRP/RANKL or sFRP/peptide motif for example SEQ ID NO: 9
  • the ability of either the sFRP or fragment thereof, or the peptide motif to bind to the test compound can be determined as above.
  • IX Incorporation ofsFRP therapeutically effective fragments, fusions, and variants of sFRP or the peptide motif into pharmaceutical compositions and methods of treatment
  • sFRP purified sFRP, sFRP fragments, sFRP variants, or peptide motifs that bind sFRP are generally combined with a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations can contain only a single peptide, or can be composed of more than one variety of sFRP fragments and/or peptide motifs.
  • the nature of the carrier will depend on the particular mode of adnrinistration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, human albumin or the like as a vehicle.
  • injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, human albumin or the like as a vehicle.
  • solid compositions e.g.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts oJ non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • protein-based pharmaceuticals can be only inefficiently delivered through ingestion.
  • pill-based forms of pharmaceutical proteins can alternatively be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow-release formulations can be produced by combining the target protein with a biocompatible matrix, such as cholesterol.
  • a biocompatible matrix such as cholesterol.
  • Another possible method of administering protein pharmaceuticals is through the use of mini osmotic pumps.
  • mini osmotic pumps As stated above a biocompatible carrier would also be used in conjunction with this method of delivery.
  • peptide motifs disclosed herein as well as sFRP could be delivered to cells in the nucleic acid form and subsequently translated by the host cell. This could be done, for example through the use of viral vectors or liposomes. Liposomes could also be used for the delivery of the protein itself.
  • the pharmaceutical compositions of the present disclosure can be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of sFRP fragments can be dete ⁇ riined readily by those with ordinary skill in the clinical art of treating conditions associated with abnormal bone remodeling. For use in treating these conditions, the described proteins are administered in an amount effective to either increase osteoclastogenesis activity or decrease osteoclastogenesis.
  • Such dosages include amounts which raise target tissue concentrations to levels at which the therapeutic activity has been observed in vitro.
  • the proteins disclosed herein can also be used to modulate T-cell interactions and immune system functions. Doses sufficient to achieve a tissue concentration that causes an increase or a decrease in osteoclastogenesis and/or T-cell activity can be determined by using the amounts described in the examples that follow.
  • the peptides or proteins can be administered to a host in vivo, such as for example, through systemic administration, such as intravenous or intraperitoneal administration. Also, the peptides or proteins can be aclministered intralesionally: i.e. the peptide or protein is injected directly into the tumor or affected area.
  • Effective doses of the disclosed peptides for therapeutic application will vary depending on the nature and severity of the condition to be treated, the age and condition of the subject and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the clinician. Typically, the dose range will be from about 0.1 ⁇ g/kg body weight to about 100 mg/kg body weight. Other suitable ranges include doses of from about 1 ⁇ g/kg to 10 mg/kg body weight.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the protein.
  • Examples of dosing schedules are 3 ⁇ g/kg acrministered twice a week, three times a week or daily; a dose of 7 ⁇ g/kg twice a week, three times a week or daily; a dose of 10 ⁇ g/kg twice a week, three times a week or daily; or a dose of 30 ⁇ g/kg twice a week, three times a week or daily.
  • a ⁇ rninister doses such as those described above by alternate routes including intravenously or intrathecally. Continuous infusion can also be appropriate.
  • Polypeptides that bind to sFRP-1 were identified using an open-ended approach. This approach involved screening a peptide phage display cDNA library for sequences that bound to recombinant sFRP-1 (Uren et al, J. Biol. CAem.275:4374-4382, 2000). Peptides that had been identified by phage display were operably linked to a sequence encoding alkaline phosphatase creating a fusion protein that, upon binding to sFRP-1, could be detected. This methodology resulted in the identification of a predominant peptide motif containing the sequence L/V-D-G-R-W-L/V
  • the sequence of the peptide motif that binds sFRP was then used to identify proteins that potentially bind to sFRP-1.
  • the RANKL protein was identified as a potential candidate because it contained an amino acid sequence that is similar to that of the peptide motif (SEQ ID NO: 9).
  • ELISA analysis using RANKL and sFRP-1 indicated that sFRP-1 binds to RANKL.
  • RANKL is known to be involved with osteoclast differentiation.
  • sFRP-1 SEQ ED NO: 3
  • a synthetic peptide containing the peptide motif enhanced osteoclastogenesis.
  • sFRP-1 Recombinant human sFRP-1 was prepared as described (Uren et al, J. Biol. Chem. 275:4374-4382, 2000).
  • the coding sequence of mouse sFRP-2 was amplified by RT-PCR, using total RNA from embryonic mouse kidney as a source, subcloned into pcDNA3.1 expression vector, transfected into MDCK cells and the recombinant protein purified by heparin-affrnity chromatography essentially as described for sFRP-1 in Uren et al, J. Biol. Chem. 275:4374-4382, 2000.
  • Rabbit polyclonal antiserum was raised against recombinant human sFRP-1 by injecting ⁇ 10 ⁇ g of purified protein with complete Freund's adjuvant into the inguinal lymph nodes, and subsequently injecting intramuscularly at 2-3 week intervals similar quantities of antigen dissolved in the incomplete Freund's adjuvant. After several boosts, an immunoglobulin fraction was obtained from serum by chromatography with protein G-bound Sepharose (Pharmacia Biotech, Uppsala, Sweden). Peptides were synthesized using standard solid phase chemistry, purified by reverse-phase
  • recombinant soluble RANKL was purchased from Peprotech, Rocky Hill, New Jersey, or residues 158-317 of murine RANKL were prepared as a GST-expressed protein.
  • M- CSF was obtained from Research Genetics Institute (Boston, MA, USA).
  • the M13 phage-displayed random peptide library was constructed as described (Adey et al. Methods in Molecular and Cellular Biology 6:3-14, 1995/1996).
  • tsJ2, tsJIO and tsJ14 were generated by transfection with a retroviral vector expressing a temperature-sensitive variant of the immortalizing gene of SV40 (ts A58; Chambers et al., Proc Natl. Acad. Sci. USA 90:5578-5582, 1993; Owens et al, Biochem. Biophys. Res. Commun. 222:225-229, 1996).
  • RAW264.7 cells were purchased from the ATCC, and the cell lines KUSA/O and mc-3T3-el are described in Horwood et al Endocrinology 139:4143-4146, 1998.
  • Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells.
  • l ,25(OH 2 ) vitamin D 3 was purchased from Wako Pure Chemicals Co. (Osaka, Japan).
  • PGE2 was obtained from Sigma (St. Louis, MO). Other chemicals and reagents were of analytical grade.
  • MDCK cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Rockville, Maryland) containing 10% fetal calf serum (Colorado Serum Company, Denver, Colorado) in 5% C0 2 at 37°C. 3. Screening of Peptide Phage Display Library
  • Amplified phage recovered from bacterial broth after this first enrichment step were subjected to two more rounds of panning in wells coated with sFRP-1 as described in the previous paragraph, except that the phage were incubated for only 2 hours and 1 hour in the second and third panning steps, respectively.
  • phage obtained from the sFRP-1- coated well were titered and seeded on a lawn of bacteria to permit isolation of phage from 200 separate colonies. Bacteria from each of these colonies were grown in broth, pelleted by centrifugation and phage retrieved in the supernatant.
  • phage supernatants were tested for binding to sFRP-1 -coated ELISA wells versus wells only coated with the BSA blocking solution. Phage were detected in this assay with primary antibody directed against phage coat protein (Pharmacia Biotech, Uppsala, Sweden, #27-9411-01) and standard detection reagents. Approximately 100 phage isolates were selected for sequence analysis, based on exhibiting at least 5- fold higher binding to sFRP-1 versus BSA coated wells.
  • sequence of the DNA insert encoding the peptide segment linked to the M13 gene III coat protein from each phage isolate was determined by using sequencing primers corresponding to adjacent vector sequence.
  • An advanced BLAST search analysis of GenBank databases was performed to identify proteins that contained sequences matching portions of the peptide sequences identified by screening of the peptide phage display library.
  • Synthetic oligonucleotides encoding peptides of interest were ligated into the bacterial alkaline phosphatase fusion vector, pMYlOl, which had been digested with Sal I aadXho I (Ya ⁇ mbhai and Kay, Anal Biochem. 247:143-151, 1997). All recombinants were confirmed by DNA sequence analysis.
  • Bacteria E.
  • coli, strain DH5 ⁇ F') transformed with the peptide/AP constructs were grown in Luria broth containing ampicillin (50 ⁇ g/mL) to an optical density of 0.5 (at 600 nm), treated with 1 mM isopropyl- ⁇ -D-thiogalactopyranoside and then incubated overnight at 37°C.
  • Conditioned medium containing peptide/AP chimera was recovered by centrifugation at 7000 g for 15 minutes. Chimeric proteins in conditioned medium were stable when stored for a few weeks at 4°C or for several months when stored at -80°C.
  • ELISA experiments were generally performed as previously described (Uren et al, J. Biol. C/zem.275:4374-4382, 2000), with modifications depending on the sFRP binding partner to be tested.
  • wells were coated with 0.5 or 1 ⁇ g of sFRP-1, blocked with BSA (0.2%, 1%, or 4%) and then incubated with putative binding partner overnight at room temperature.
  • BSA 0.2%, 1%, or 48%
  • putative binding partner overnight at room temperature.
  • pNPP p-nitrophenolphosphate
  • soluble peptides were preincubated with peptide/AP chimeras in bacterial broth for 30 min at room temperature prior to transfer into ELISA wells coated with sFRP-1 or BSA.
  • serial dilutions of soluble RANKL were assayed in replicate.
  • RANKL solutions were aspirated and bound RANKL was detected by sequential incubations with primary antibody to RANKL, secondary antibody coupled with AP and pNPP.
  • Similar experimental designs were employed when other TNF ⁇ family members were examined for binding to sFRPs, and when sFRP-1 derivatives or sFRP-2 were the binding targets for RANKL.
  • TTC Titration Calorimetry
  • ddPCR was performed essentially as described (Liang et al, Science 257:967-971, 1992 and Schwarzedes et al., J. Biol. Chem. 270:20891-20894, 1995), except 1 ⁇ g of total RNA was reverse transcribed.
  • PCR products were cloned into pCRScriptll (Stratagene, LaJolla, CA) or pGEM-T (Promega, Madison, WI). DNA sequence analysis was performed using a T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden). Oligonucleotides were synthesized on an Oligo 1000M DNA Synthesizer (Beckman Instruments Inc., Fullerton CA, USA). The oligonucleotides were: for ddPCR, DDMR-2 (5'-CTTGATTGCC-3'; SEQ ID NO: 37) and T12VA (5-TTTTTTTTTTTTTTTT[A,C,G]A; SEQ ID NO: 32-3').
  • Partial cDNA fragments were amplified using 5'-10mers resulting in the synthesis of varying length cDNAs due to random annealing to different reverse transcribed mRNA species. This PCR reaction is performed at an annealing temperature of 40°C and in the presence of [ ⁇ 35 S]-dATP to allow the visualization of resulting products.
  • PCR products were resolved on 6% polyacrylamide sequencing gels and exposed to X-ray film for 1-3 days.
  • Differentially regulated cDNA fragments were excised from the gel by overlaying the film and cutting out the region of interest.
  • the cDNA fragment was reamplified by two rounds of PCR (a total of 80 cycles of PCR).
  • the reamplified product was then molecularly cloned into pGEM-T (Promega Inc., Madison, Wisconsin), and the nucleic acid sequence ofthe amplified insert was determined.
  • RNA isolated from cell lines or tissues was reverse transcribed with oligo-dT and PCR performed with the primers sfrp-la (5'-TTAAAATTGCTGCCTGCCTGAG-3'; SEQ ID NO: 38) and sfrp-lb (5'-TCCGAACTACAGGGACAACAGG-3'; SEQ ID NO: 39) for 22 cycles, which was found to be in the log-linear phase of amplification for sFRP-1 transcripts from osteoblastic sources. Amplifications were performed according to manufacturer's instructions.
  • PCR products were electrophoresed, transferred to nylon membrane, and hybridized with ⁇ - 32 P-labeled internal detection oligonucleotide, sfrp-lc (5'-GCCCAGAGGTATTTCTCAAAGTTG-3'; SEQ ID NO: 39).
  • gapdh-2 (5'-ATGAGGTCCACCACCCTGTT-3'; SEQ ID NO: 33, nucleotides 640-659; Tso et al, Nucl. Acids Res.
  • gapdh-4 were used to amplify the normalizing gene, glyceraldehyde-3 -phosphate dehydrogenase, by 20 cycles of PCR and products were detected with ⁇ - 32 P-labeled gapdh-1 as described (Suda et ⁇ /., J. Cell. Physiol 166:94-104, 1996).
  • a murine sFRP-1 riboprobe was generated by PCR using RNA derived from tsJ2 cells. The resultant fragment of 750 bp was cloned into pGEM-T (Promega, Madison, WI, USA). The plasmid was linearized and transcribed with T7 or SP6 RNA polymerase to generate antisense or sense riboprobes. The riboprobes were labeled with digoxigenin (DIG) during RNA transcription using a RNA labeling kit (Boehringer Mannheim, Mannheim GmbH, Germany) according to the manufacturer's instructions. In situ hybridization was performed as previously described (Kartsogiannis et al, Bone 21:385-392, 1997).
  • DIG digoxigenin
  • Osteoblastic cells were prepared from the calvaria of newborn mice by digestion with 0.1% collagenase (Worthington Biochemical Co., Freefold, Australia) and 0.2% dispase (Godo Shusei, Tokyo, Japan). Bone marrow and spleen cells were obtained from adult and from newborn mice, respectively (Udagawa et al, J. Exp. Med. 182: 1461-1468, 1995). Osteoblastic cells were co- cultured with bone marrow or spleen cells as described previously (Udagawa et al, J. Exp. Med. 182: 1461-1468, 1995,).
  • primary osteoblastic cells (2 x 10 /well) and nucleated spleen cells (1 x 10 6 /well) or marrow cells (5 x 10 5 /well) were co-cultured in 48-well plates (Corning Glass Inc., Corning, NY) with 0.4 mL of ⁇ -MEM (GIBCO/BRL, Grand Island, NY) containing 10% fetal bovine serum (Cytosystems, Castle Hill, NSW, Australia) in the presence of test chemicals. Cultures were incubated in quadruplicate and cells were replenished on day 3 with fresh medium. Osteoclast formation was evaluated after culturing for 6-7 days.
  • Adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), and the number of TRAP -positive osteoclasts was scored as described (Udagawa et al, J. Exp. Med. 182: 1461-1468, 1995).
  • TRAP staining adherent cells were fixed with 4% formaldehyde in PBS for 3 minutes.
  • experiments were performed either with adult mouse spleen cells or with RAW264.7 cells treated with M-CSF and RANKL as described in Quinn et al, Endocrinology 139:4424-4427, 1998. Where indicated, these assays were conducted in the presence or absence of splenic T cells. T cell fractions were prepared as described in Horwood et al, Journal of Clinical Investigation 101:595-603, 1998.
  • Example 2 Identification of Peptides that bind sFRP-1
  • ⁇ 25 x 10 9 phages from a library containing a diverse repertoire of twelve-amino acid residue segments linked to the gene III coat protein of Ml 3 phage were screened.
  • the phage preparation selected for its ability to bind sFRP-1 was titered and then plated on a lawn of bacteria. Phage from 200 separate colonies of lysed bacteria were picked, grown in bacterial broth overnight, recovered in supernatant, and tested for their ability to bind preferentially to sFRP-1 versus BSA in an ELISA. Phage that bound at least five times more avidly to sFRP-1 than BSA-coated wells were subjected to nucleotide sequence analysis to determine the identity of the peptide sequence responsible for this binding specificity.
  • a set of peptide-alkaline phosphatase fusion proteins containing the peptide motifs were generated. These fusion proteins were tested for specific binding to sFRP-1 (SEQ ID NO: 3) in an ELISA format. As illustrated in FIG. 1, broths from multiple isolates of the A-C2 (SEQ IN NO: 14)/alkaline phosphatase fusion protein all showed strong, highly specific binding to wells preincubated with sFRP-1. Similar results were obtained with the A-E4 (SEQ ID NO: 1 l)/alkaline phosphatase fusion protein.
  • the B-B9 (SEQ ID NO: 27) /alkaline phosphatase fusion protein did not exhibit specific binding to sFRP-1.
  • This qualitative difference between A-C2 (SEQ ID NO: 14), A-E4 (SEQ ID NO: 11), and B-B9 (SEQ ID NO: 27) derivatives was consistent with a quantitative difference noted during the ELISA screening of the respective phage.
  • the A-C2- (SEQ ED NO: 14) and A-E4- (SEQ ID NO: 11) expressing phage were more abundant in the phage preparation selected for sFRP-1 binding (Table 1) and showed a higher ratio of sFRP-1 :BSA binding than B-B9 (SEQ ID NO: 27) phage.
  • each phage particle has five copies of the peptide displayed on its surface, whereas the peptide-alkaline phosphatase fusion proteins exist as dimers in solution.
  • the relatively weaker binding avidity of the B-B9 sequence as originally perceived with the pentavalent phage particle became more obvious when dimeric reagents were tested.
  • ITC Isothermal titration calorimetry
  • A-D9 Another peptide, A-D9, was analyzed in a manner similar to the routine followed for A-C2.
  • ELISA experiments performed with an A-D9/AP chimera showed that this chimera bound specifically to wells coated with sFRP-1 rather than BSA. This binding was blocked in a dose-dependent manner with soluble synthetic peptide containing the A-D9 sequence. Binding of the A-D9/AP chimera to sFRP-1 in ELISA wells was disrupted by alanine substitutions in the A-D9 sequence.
  • A-C2 peptide also could inhibit binding of the A-D9/AP chimera to sFRP-1 and the A-D9 peptide inhibited binding of the A-C2/AP chimera to sFRP-1.
  • DGR common element
  • the sequence V-V-D-G-R-F-V (SEQ ED NO: 10) in the human atrial natriuretic peptide (ANP) receptor A is also of significance because of the co-expression of this gene product and sFRP-1 in tissues within the kidney and eye.
  • sFRP-1 SEQ ID NO: 3
  • RANKL interact with each other in a manner that has significant biological consequences, and their interaction can be modulated to affect osteoclastogenesis.
  • Example 5 sFRP-1 and Expression in Bone In situ hybridization analyses of sFRP-1 transcripts (SEQ ID NO: 1) in skeletal structures of mouse embryos (Day 19), newborn mice (Day 1) and adult mice (five weeks) were performed to examine the role of sFRP-1 in bone development.
  • Hypertrophic chondrocytes were strongly positive in murine embryos (E19).
  • E19 murine embryos
  • spinal cord of Day 1 mice there was very strong expression in the ossification center within the cartilage primordium of the lumbar vertebral body and the nucleus pulposus in the central part of the lumbar invertebral disc.
  • bone lining cells were positive as well as a number of isolated marrow cells, and osteocytes were weakly positive.
  • sFRP-1 mRNA was also observed in the epidermis. RANKL is expressed in a similar pattern (Kartsogianms et al, Bone 25:525-534, 1999). Expression of sFRP-1 in skeletal sites was also detected. Hence, it is likely that sFRP-1 is involved in skeletal morphogenesis and sFRP-1 expression continues in a number of sites through to adulthood. sFRP-1 expression in osteoblasts (tsJJ2 cells) was studied (for a description of the tsJJ2 cell line and the tsJ14 cell line see Chambers et al, Proc. Natl. Acad. Sci. USA 90:5578-5582, 1993).
  • sFRP-1 is preferentially expressed in osteoblasts (tsJ2 cells) that promote osteoclast formation.
  • Murine sFRP-1 transcripts were amplified using the oligonucleotides sfrp-la and sfip-lb. Amplified products were verified by Southern analysis using [ ⁇ - 32 P]dATP end-labeled oligonucleotide sfrp-lb as a probe.
  • Differential display PCR also showed that sFRP-1 is upregulated in osteoblast lines that stimulate osteoclastogenesis, but not in the products from two other lines that do not support osteoclast differentiation.
  • osteotropic factors such as l ⁇ ,25(OH 2 ) vitamin D 3 caused limited stimulation of sFRP-1 expression by osteoblastic lines.
  • Total RNA was isolated from either untreated or cells treated with l ⁇ ,25(OH 2 ) vitamin D 3 for 24 hours, reverse transcribed with oligo (dT), and subjected to PCR for murine SFRP1 and GAPDH.
  • a co-culture of osteoblasts and bone marrow treated for 24 hours with l ⁇ ,25(OH 2 ) vitamin D 3 was included as a positive control.
  • the primer combination of sfrp-la (5'- AGC CTT GGC AGT CAA CGA CG-3' SEQ ID NO: 30) and sfrp-lb (5'- GTT GTG GCT TTT GCA TTG CAC-3' SEQ ID NO: 31) was used for sFRP-1 amplification and the primer combination of gapdh-2 (5'-ATG AGG TCC ACC ACC CTG TT-3' SEQ ID NO: 33) and gapdh-4 (5'-CAT GGA GAA GGC TGG GGC TC-3' SEQ ID NO: 34) was used for GAPDH amplification.
  • PCR products were electrophoresed, transferred to nylon membrane and hybridized with [ ⁇ - 32 P]-labeled internal detection oligonucleotide, sfrp-lc (5'-TGT TGA AAA CTA GTA GCT G-3' SEQ ID NO: 35) and gapdh-1 (5'-GCT GTG GGC AAG GTC ATC CC-3' SEQ ID NO: 36), respectively, as described (Southby et al, Endocrinology 137:1349-1357, 1996).
  • RT-PCR analysis was repeated in triplicate. Semiquantitative RT-PCR analysis was performed three times on each RT reaction and two independent RT reactions were examined.
  • sFRP-1 may be a mediator of hormonally dependent osteoclast formation.
  • sFRP-1 expression increased markedly when osteoblasts and osteoclast progenitors were co-cultured. The time course of this increase matched the rise in appearance of TRAP+ cells, a marker of osteoclast differentiation.
  • upregulation of sFRP-1 expression is dependent on cell-cell communication between the osteoblast and osteoclast lineages.
  • the correlation between sFRP-1 expression and osteoclast formation suggested that sFRP-1 induction might be a consequence of osteoclastogenesis.
  • Example 6 sFKP-l Blocks Osteoclastogenesis in Cell Culture Bioassays
  • the possibility that sFRP-1 (SEQ ID NO: 3) and RANKL interact directly with each other was tested using an ELISA assay.
  • the ELISA assay involved the use of wells that were coated with recombinant sFRP-1 (SEQ ID NO: 3) and subsequently blocked with BSA.
  • RANKL was then incubated in these wells and in adjacent wells that had only been treated with BSA. Subsequent detection with RANKL antiserum and secondary reagents revealed that RANKL bound specifically to sFRP-1 (FIG. 5). This result was confirmed in several separate experiments.
  • TGF ⁇ was added during the first three days of culture to increase osteoclast numbers (FIG. 14).
  • sFRP-1 inhibited TNF ⁇ -dependent osteoclast formation when present during the first three days of culture, whilst OPG had no effect suggesting that sFRP-1 was acting indirectly of RANKL, through binding to TNF ⁇ or through WNT signaling.
  • the effect of bacterially expressed CRD was assessed in three different cell culture models of osteoclast formation. These were: (1) bone marrow cells + RANKL + M-CSF, (2) the macrophage/monocyte cell line RAW264.7 + RANKL, and (3) RAW264.7 + TNF ⁇ + TGF ⁇ (Horwood et al, Journal of Immunology 166:4915-4921, 2001; Quinn et al, Journal of Bone and Mineral Research. 16, 1787-1794, 2001). In each system, both RANKL-dependent (cultures 1 and 2) and RANKL-independent (culture 3, TNF ⁇ -dependent osteoclast formation), the bacterially expressed CRD mimicked the action of full-length sFRP-1 and with similar potency (FIG. 15).
  • sFRP-1 reduced the number of multinucleated TRAP+ cells by 50% when used at a concentration 300 ng/mL, while a dose of 1.6 ug/mL decreased the number of cells by 95% (FIG. 6A).
  • a similar dose-response pattern was observed when adult mouse spleen cells were treated with RANKL and M-CSF (FIG. 6B).
  • A-C2 Synthetic Peptide Promotes Osteoclast Formation
  • A-C2 (SEQ ED NO: 14) polypeptide has sequence homology to RANKL and because A-C2 (SEQ ID NO: 14) binds to sFRP-1, assays were performed to determine if A-C2 (SEQ ID NO: 14) would block sFRP-1 binding to RANKL and thus increase osteoclastogenesis. Consistent with this hypothesis, treatment of osteoblast and adult spleen cell co-cultures with A-C2 (SEQ ID NO: 14) resulted in a ten-fold increase in TRAP+ multinucleated cells (FIG. 8).
  • A-C2 (SEQ ED NO: 14) only had an effect on osteoclast formation when it was present during day 0 to day 3 of the co-culture experiment. RANKL presence is also required for osteoclastogenesis during day 0 to day 3 (Suda et al, Endocrine Reviews 20:345-357, 1999). Thus, these results are consistent with the notion that A-C2 (SEQ ID NO: 14) most likely has an impact on RANKL activity.
  • A-C2 SEQ ID NO: 14
  • A-C2 SEQ ID NO: 14
  • FIG. 11 A-C2 (SEQ ID NO: 14) dose-dependent stimulation of TRAP+, multinucleated cell differentiation in RAW264.7 cultures was only observed when T cells were added to the cultures.
  • Example 9 Identification of a Biologically Relevant Peptide Motif The peptide motif, L/V-V-D-G-R-W-L V (SEQ ID NO: 9), has not been previously identified as being capable of binding to sFRP-1.
  • This binding activity has been characterized through the use of a series of ELISA experiments using peptide/alkaline phosphatase fusion proteins, alanine scanning mutagenesis and synthetic peptides. These experiments also demonstrated that substitutions in nearby residues could enhance or reduce the peptide motif/sFRP-1 binding, implying that systematic substitutions in adjacent residues and conservative changes within the peptide motif, are able to strengthen the interaction with sFRP-1.
  • Conservative substitutions replace one amino acid with another amino acid that is similar in size, hydrophobicity, etc. Such substitutions generally are conservative when it is desired to finely modulate the characteristics of the protein.
  • amino acids which canbe substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; He or Val for Leu; Arg or Gin for Lys; Leu or He for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and He or Leu for Val.
  • the binding affect of peptides in which such substitutions have been made can readily be coiifirmed by the peptide motif-binding assay disclosed herein.
  • substitutions that are less conservative than those described above, i.e. selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histadyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • nucleic acid sequences encoding the peptide motif can be designed by either synthetically synthesizing the appropriate nucleic acid sequence, or by using PCR to amplify the appropriate sequence. Once obtained the nucleic acid sequence can be placed in an appropriate expression vector and transformed into an organism such that the organism then produces the peptide.
  • the significance of the peptide motif (SEQ ED NO: 9) was demonstrated in experiments that revealed a dramatic stimulatory effect of a synthetic peptide bearing the peptide motif in osteoclastogenesis bioassays.
  • the ability of the A-C2 peptide (SEQ TD NO: 14) to promote osteoclast formation was consistent with the idea that the peptide increased RANKL activity by blocking the inhibitory effect of endogenous sFRP-1 (FIG. 17).
  • the requirement of T cells for the A-C2 peptide (SEQ ED NO: 14) to stimulate osteoclast formation implied that splenic T cells express an osteoclastogenesis inhibitor sensitive to A-C2 (SEQ ED NO: 14). This inhibitor is believed to be sFRP-1, as sFRP-1 is expressed in spleen (Finch et al, Proc. Natl Acad. Sci. USA 94:6770-6775, 1997).
  • reagents possessing a sFRP-1 binding motif are believed to have utility in regulating RANKL signaling involved in T cell-dendritic communication that is modulated by endogenous sFRP-1.
  • Such regulation can be exploited to optimize vaccine therapies in a variety of settings where T cell-dendritic cell interactions have an important role in the immune response.
  • Dendritic cells have been shown to promote vaccine responses, which can be determined by measuring titers to known antigens developed in inoculated animals (M. Di Nicola et al. , Cytokines Cell. Mol. TJier. 4: 265-273, 1998; C. Reis e Sousa et al, Curr. Opin. Immunol. 11: 392-399, 1999; K. Tarte and B. Klein, Leukemia 13: 653-663, 1999). Efforts are underway to optimize the expansion of immunologically responseive dendritic cells in order to improve the efficacy of vaccine therapy (R. Hajek and A.W. Butch, Med. Oncol. 17: 2-15, 2000). Using methods cited in the above references, reagents corresponding to the sFRP-1 binding motif and sFRP-1 canbe useful to enhance the immune response in vaccine therapies.
  • sFRP-1 can be useful for modulating endogenous proteolytic cleavage of RANKL.
  • a large fraction of the RANKL expressed by T cells is proteolytically processed to release a soluble, biologically active form. This process, which can be mediated by the TNF converting enzyme (TACE), involves cleavage at one or two sites in the TNF converting enzyme (TACE).
  • TACE TNF converting enzyme
  • RANKL sequence just upstream of the putative peptide-binding motif (SEQ ID NO: 9). Therefore, it is believed that sFRP-1 regulates the proteolytic processing of RANKL in a manner that could be reversed by reagents containing the peptide motif (SEQ ID NO: 9).
  • sFRP-1 ability of sFRP-1 to regulate RANKL processing is tested by culturing T-cells expressing RANKL and treating the cultures with various concentrations of sFRP-1. The resulting soluble RANKL proteins are then identified using a RANKL specific western blot. The degree of RANKL processing is then correlated to the sFRP concentration in the sample.
  • reagents containing the peptide motif also have utility in disrupting the interaction of sFRP-1 with other proteins.
  • other known proteins like the netrin receptor, and UNC5H3 and the ANP receptor A, have sequences similar to the binding motif, and newly identified gene sequences can be routinely screened for such sequences. Proteins with this motif are likely to be additional potential binding partners for sFRP-1 and targets for reagents containing the sFRP-1 binding motif. For instance, sFRP-1 binding to the ANP receptor A could regulate the release of sodium and fluid in the kidney and eye.
  • sFRP-1 has an inhibitory effect on osteoclastogenesis, which is likely due to its interaction with RANKL.
  • the elevated expression of sFRP-1 transcript specifically in osteoblast lines capable of stimulating osteoclast formation initially suggested that sFRP-1 promotes osteoclastogenesis.
  • the increase in sFRP-1 transcript observed in co-cultures of osteoblasts and hematopoietic progenitors as osteoclast formation proceeded implied that sFRP-1 was instead part of a tonic mechanism to limit the extent of osteoclast formation (FIG. 17).
  • sFRP-1 can have clinical utility in conditions where excessive osteoclast activity has pathological consequences. Osteoporosis and hypercalcemic osteopaenia are exampls of such conditions; rheumatoid arthritis is another, which could be a particularly good target for sFRP-1 therapy because soluble RANKL from T cells is thought to have an important role in the bone loss associated with this disease.
  • TNF-ligand family members are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes.
  • Cellular responses to TNF-family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis.
  • Apoptosis-programmed cell death is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes.
  • Diseases associated with increased cell survival, or the inhibition of apoptosis include cancers, autoimmune disorders, viral infections, inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
  • Diseases associated with increased apoptosis include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock cachexia and anorexia.
  • the disclosure further provides methods for modulating the TNF ligand/TNF receptor interactions. These methods involve contacting sFRP, a fragment or variant of sFRP, or the peptide motif (SEQ ID NO: 9) with a member of the TNF-ligand family of proteins, and detecting a change in TNF-ligand biological activity.
  • sFRP sFRP
  • fragment or variant of sFRP or the peptide motif acts as an "agonist” or antagonist”
  • TNF-family ligand/receptor cellular response assays such as ones described in the references cited in the following reviews: D. Wallach et al, Annu. Rev. Immunol. 17: 331-367, 1999; S.J. Baker and E.P. Reddy, Oncogene 17: 3261-3270, 1998
  • the disclosure provides screening methods for dete ⁇ riining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Rheumatology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pain & Pain Management (AREA)
  • Obesity (AREA)
  • Neurology (AREA)

Abstract

Cette invention concerne un motif de peptide et des protéines renfermant ledit motif qui peuvent se lier à des éléments de la famille de protéine sécrétée Frizzled (sFRP). Ladite invention a également trait à des méthodes de régulation de l'interaction de sFRP-1 avec des protéines contenant ledit motif.
PCT/US2002/000869 2001-01-10 2002-01-10 Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes WO2002055547A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU2002241859A AU2002241859B2 (en) 2001-01-10 2002-01-10 sFRP and peptide motifs that interact with sFRP and methods of their use
ES02707454T ES2385666T3 (es) 2001-01-10 2002-01-10 SFRP y motivos peptídicos que interactúan con SFRP y sus métodos de uso
US10/466,136 US7488710B2 (en) 2001-01-10 2002-01-10 SFRP and peptide motifs that interact with SFRP and methods of their use
DK02707454.1T DK1387854T3 (da) 2001-01-10 2002-01-10 SFRP og peptidmotiver som interagerer med SFRP og fremgangsmåder til anvendelse deraf
CA2434672A CA2434672C (fr) 2001-01-10 2002-01-10 Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes
EP02707454A EP1387854B1 (fr) 2001-01-10 2002-01-10 Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes
JP2002556615A JP4029041B2 (ja) 2001-01-10 2002-01-10 ｓＦＲＰおよびｓＦＲＰと相互作用するペプチドモチーフならびにそれらの使用法
AT02707454T ATE550345T1 (de) 2001-01-10 2002-01-10 Sfrp, mit sfrp-interagierende peptidmotive sowie verwendungsverfahren
US12/019,567 US8334260B2 (en) 2001-01-10 2008-01-24 sFRP and peptide motifs that interact with sFRP and methods of their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26090801P 2001-01-10 2001-01-10
US60/260,908 2001-01-10

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10466136 A-371-Of-International 2002-01-10
US12/019,567 Division US8334260B2 (en) 2001-01-10 2008-01-24 sFRP and peptide motifs that interact with sFRP and methods of their use

Publications (3)

Publication Number Publication Date
WO2002055547A2 WO2002055547A2 (fr) 2002-07-18
WO2002055547A9 true WO2002055547A9 (fr) 2002-10-31
WO2002055547A3 WO2002055547A3 (fr) 2003-11-20

Family

ID=22991151

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/000869 WO2002055547A2 (fr) 2001-01-10 2002-01-10 Sfrp et motifs de peptides interagissant avec sfrp et methodes d'utilisation correspondantes

Country Status (11)

Country Link
US (2) US7488710B2 (fr)
EP (1) EP1387854B1 (fr)
JP (3) JP4029041B2 (fr)
AT (1) ATE550345T1 (fr)
AU (1) AU2002241859B2 (fr)
CA (1) CA2434672C (fr)
CY (1) CY1112875T1 (fr)
DK (1) DK1387854T3 (fr)
ES (1) ES2385666T3 (fr)
PT (1) PT1387854E (fr)
WO (1) WO2002055547A2 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE602005023286D1 (de) * 2004-03-31 2010-10-14 Kazuo Shinya Verfahren zum biosubstanzeinfang, zur strukturanalyse oder/und identifizierung unter verwendung einer markierungssubstanz
EP1831254B1 (fr) 2004-12-23 2012-06-06 Molmed SpA Produit de conjugaison
WO2008061029A1 (fr) * 2006-11-10 2008-05-22 Wyeth Dérivés arylsulfonamide et procédés de leur utilisation
AU2009226161A1 (en) * 2008-03-21 2009-09-24 Adlyfe, Inc. Use of pyrene to carry peptides across the blood brain barrier
EP2293685B1 (fr) * 2008-05-16 2015-04-29 Siebte PMI Verwaltungs GmbH Concentrés et isolats protéiques d'oléagineux, et leurs procédés de production
JP2013006783A (ja) * 2011-06-22 2013-01-10 Nagasaki Univ 荷重感知遺伝子
EP3116521B1 (fr) * 2014-03-14 2024-07-31 The Research Foundation for The State University of New York Régulation neurogène de la croissance osseuse et de la dégradation osseuse
PL3139979T3 (pl) 2014-05-07 2023-12-27 Boehringer Ingelheim International Gmbh Jednostka, nebulizator i sposób
US20200216881A1 (en) * 2017-06-15 2020-07-09 Technion Research & Development Foundation Limited Compositions and methods for detection of genomic variations
EP3696192A1 (fr) * 2019-02-15 2020-08-19 Consejo Superior De Investigaciones Científicas Cible thérapeutique et anticorps monoclonaux contre elle pour le diagnostic et le traitement de la maladie d'alzheimer

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5238839A (en) 1988-02-09 1993-08-24 Dana Farber Cancer Institute Nucleic Acids Encoding proteins which induce immunological effector cell activation and chemattraction, vectors, and recombinant cells
US5204328A (en) 1990-06-26 1993-04-20 Merck & Co., Inc. Peptides having atrial natriuretic factor activity
AU3141693A (en) 1991-11-22 1993-06-15 Friedman, Mark M. Non-peptidic surrogates of the arg-gly-asp sequence and pharmaceutical compositions comprising them
US5578569A (en) 1993-03-12 1996-11-26 Tam; Cherk S. Method of increasing bone growth
US5536814A (en) 1993-09-27 1996-07-16 La Jolla Cancer Research Foundation Integrin-binding peptides
US5846932A (en) 1993-11-12 1998-12-08 Genentech, Inc. Receptor specific atrial natriuretic peptides
US5726156A (en) 1995-03-06 1998-03-10 Trega Biosciences, Inc. Cytokine regulatory agents and methods of use in pathologies and conditions associated with altered cytokine levels
US5632983A (en) 1994-11-17 1997-05-27 University Of South Florida Method for treating secondary immunodeficiency
US5861483A (en) 1996-04-03 1999-01-19 Pro-Neuron, Inc. Inhibitor of stem cell proliferation and uses thereof
WO1997039357A1 (fr) 1996-04-12 1997-10-23 The Board Of Trustees Of The Leland Stanford Junior University COMPOSITIONS A BASE DU RECEPTEUR DE Wnt ET PROCEDES D'UTILISATION DE CELLES-CI
US5851984A (en) 1996-08-16 1998-12-22 Genentech, Inc. Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides
JP4299886B2 (ja) 1996-09-24 2009-07-22 タノックス インコーポレイテッド アポトーシス関連ペプチドをコードする遺伝子ファミリー、それによってコードされるペプチド、およびそれらの使用方法
US5939271A (en) 1997-02-19 1999-08-17 The Regents Of The University Of California Netrin receptor
RO128635A2 (ro) 1997-04-16 2013-07-30 Amgen Inc. Anticorp sau fragment al acestuia care se leagă la opgbp şi utilizarea acestuia, utilizarea unei forme solubile de odar, şi metodă de identificare a unui compus care descreşte activitatea opgbp
EP0887408A1 (fr) 1997-05-23 1998-12-30 Smithkline Beecham Plc Oncogène Wnt-3, Polypeptides et polynucleotides
US6479255B1 (en) 1997-05-29 2002-11-12 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Polynucleotides encoding human FRP and fragments thereof
WO1999009152A1 (fr) 1997-08-12 1999-02-25 Human Genome Sciences, Inc. Proteine humaine hflp
AU1535899A (en) 1997-11-25 1999-06-15 Millennium Pharmaceuticals, Inc. Genes encoding frizzled-like proteins
WO1999029865A2 (fr) 1997-12-12 1999-06-17 The Rockefeller University Proteine appartenant a la superfamille des tnf, acides nucleiques la codant et ses methodes d'utilisation
JPH11196864A (ja) 1998-01-08 1999-07-27 Deinabekku Kenkyusho:Kk 破骨細胞の分化を誘導する方法
US6110718A (en) * 1998-03-20 2000-08-29 Wayne State University Mammalian putative phosphatidylinositol-4-phosphate-5-kinase
WO2001057188A2 (fr) 2000-02-03 2001-08-09 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
NZ529560A (en) * 1999-09-13 2005-05-27 Wyeth Corp Pharmaceutical compositions and methods of using secreted frizzled related protein
US6600018B1 (en) * 2000-04-10 2003-07-29 The United States Of America As Represented By The Department Of Health And Human Services Secreted frizzled related protein, sFRP, fragments and methods of use thereof
JP2002181893A (ja) * 2000-12-11 2002-06-26 Mitsubishi Electric Corp 半導体装置の検査方法および検査装置

Also Published As

Publication number Publication date
DK1387854T3 (da) 2012-07-09
PT1387854E (pt) 2012-06-26
JP4029041B2 (ja) 2008-01-09
US7488710B2 (en) 2009-02-10
JP4248583B2 (ja) 2009-04-02
CA2434672A1 (fr) 2002-07-18
ATE550345T1 (de) 2012-04-15
WO2002055547A3 (fr) 2003-11-20
EP1387854A2 (fr) 2004-02-11
ES2385666T3 (es) 2012-07-30
JP4248600B2 (ja) 2009-04-02
JP2007300920A (ja) 2007-11-22
US8334260B2 (en) 2012-12-18
JP2009005704A (ja) 2009-01-15
US20040077828A1 (en) 2004-04-22
JP2005501508A (ja) 2005-01-20
WO2002055547A2 (fr) 2002-07-18
US20080145884A1 (en) 2008-06-19
CA2434672C (fr) 2014-03-25
AU2002241859B2 (en) 2007-07-19
EP1387854B1 (fr) 2012-03-21
CY1112875T1 (el) 2016-04-13

Similar Documents

Publication Publication Date Title
US8334260B2 (en) sFRP and peptide motifs that interact with sFRP and methods of their use
JP2022044589A (ja) ＡｃｔＲＩＩＢから誘導されたバリアントおよびその使用
EP2401293B1 (fr) Ligands de conception de la superfamille de tgf-bêta
US20040014209A1 (en) Compositions and methods for modulating cell differentiation
JP4530129B2 (ja) スーパー抗原
AU2002241859A1 (en) sFRP and peptide motifs that interact with sFRP and methods of their use
EP2217618A2 (fr) Nouveaux gènes marqueurs de lymphocytes t régulateurs issus de sang humain
US8735355B2 (en) Methods of use of fragments of secreted frizzled related protein, sFRP
US6432410B1 (en) Morphogenic proteins
WO1996000240A1 (fr) Nouvelle proteine et procede pour sa fabrication
CN115515618A (zh) 用于治疗肾脏疾病或病症的单臂ActRIIA和ActRIIB异多聚体和方法
KR100885545B1 (ko) Ｒａｎｋ 저해 활성을 갖는 폴리펩티드, 그를 포함하는약학적 조성물, 그를 이용한 개체의 파골세포 분화를억제하는 방법 및 파골세포의 분화 및 활성화로 인하여야기되는 질병을 치료 또는 예방하는 방법, 그를 코딩하는핵산, 상기 핵산을 포함하는 벡터 및 숙주세포
KR20190025569A (ko) Tgf-베타 슈퍼패밀리의 ab6 패밀리 디자이너 리간드
WO2004035627A1 (fr) Modulation de parcours de signalisation de type beta tgf
WO1998037195A9 (fr) Proteines morphogeniques
KR20190005240A (ko) Tgf-베타 슈퍼패밀리의 ab6 패밀리 디자이너 리간드를 포함하는 골 및 연골 질환 치료용 조성물 및 그의 이용
WO1998037195A1 (fr) Proteines morphogeniques
Martin GDF9 and BMP15: species difference and synergistic interactions.

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/16-16/16, DRAWINGS, REPLACED BY NEW PAGES 1/16-16/16; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

Free format text: PAGES 1/16-16/16, DRAWINGS, REPLACED BY NEW PAGES 1/16-16/16; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWE Wipo information: entry into national phase

Ref document number: 2002241859

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2434672

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 10466136

Country of ref document: US

Ref document number: 2002556615

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2002707454

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 2002707454

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 2002241859

Country of ref document: AU